Methods and materials for producing 7-carbon monomers

ABSTRACT

This document describes biochemical pathways for producing 7-aminoheptanoic acid using a β-ketoacyl synthase or a β-ketothiolase to form an N-acetyl-5-amino-3-oxopentanoyl-CoA intermediate. 7-aminoheptanoic acid can be enzymatically converted to pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol or corresponding salts thereof. This document also describes recombinant microorganisms producing 7-aminoheptanoic acid as well as pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol or corresponding salts thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/263,299, filed Dec. 4, 2015, the contents of which are incorporatedherein by reference.

TECHNICAL FIELD

This invention provides methods for biosynthesizing 7-carbon monomers.For example, the present invention provides methods for makingN-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof using apolypeptide having the activity of a β-ketoacyl synthase or aβ-ketothiolase and enzymatically convertingN-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof to7-aminoheptanoic acid or a salt thereof using one or more polypeptideshaving the activity of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoAhydratase, a trans-2-enoyl-CoA reductase, a β-ketothiolase, athioesterase or a CoA-transferase and a deacetylase or methods usingmicroorganisms expressing one or more of such enzymes. This inventionalso provides methods for converting 7-aminoheptanoic acid to one ormore of pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and1,7-heptanediol or the corresponding salts thereof using one or morepolypeptides having the activity of isolated enzymes such asdehydrogenases, reductases, acetyltransferases, deacetylases, andtransaminases or methods using recombinant microorganisms expressing oneor more such enzymes.

BACKGROUND

Nylons are synthetic polymers composed of polyamides which are generallysynthesized by the condensation polymerization of a diamine with adicarboxylic acid. Similarly, nylons also may be produced by thecondensation polymerization of lactams. Nylon 7 is produced bypolymerization of 7-aminoheptanoic acid, whereas Nylon 7,7 is producedby condensation polymerization of pimelic acid andheptamethylenediamine. No economically cost competitive petrochemicalroute exists to produce the monomers for Nylon 7 and Nylon 7,7.

Given no economically cost competitive petrochemical monomer feedstock,biotechnology offers an alternative approach via biocatalysis.Biocatalysis is the use of biological catalysts, such as enzymes, toperform biochemical transformations of, for example, bioderivedfeedstocks and petrochemical feedstocks which can both be viablestarting materials for the biocatalysis processes.

SUMMARY

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of 7-aminoheptanoate,pimelic acid, 7-hydroxyheptanoate, heptamethylenediamine, and1,7-heptanediol or derivatives thereof, wherein the methods arebiocatalyst based. This document is based at least in part on thediscovery that it is possible to construct biochemical pathways forusing, inter alia, a polypeptide having the activity of a β-ketoacylsynthase or a β-ketothiolase to produce 7-aminoheptanoate or a saltthereof, which can be converted in one or more enzymatic steps topimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or1,7-heptanediol or corresponding salts thereof. Pimelic acid andpimelate, 7hydroxyheptanoic acid and 7-hydroxyheptanoate, and7-aminoheptanoic acid and 7-aminoheptanoate are used interchangeablyherein to refer to the compound in any of its neutral or ionized forms,including any salt forms thereof. It is understood by those skilled inthe art that the specific form will depend on pH.

For compounds containing carboxylic acid groups such as organicmonoacids, hydroxyacids, amino acids and dicarboxylic acids, thesecompounds may be formed or converted to their ionic salt form when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. The saltcan be isolated as is from the system as the salt or converted to thefree acid by reducing the pH to below the pKa through addition of acidor treatment with an acidic ion exchange resin.

For compounds containing amine groups such as but not limited to organicamines, amino acids and diamine, these compounds may be formed orconverted to their ionic salt form by addition of an acidic proton tothe amine to form the ammonium salt, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid or muconic acid. Acceptable inorganic bases are knownin the art and include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. The saltcan be isolated as is from the system as a salt or converted to the freeamine by raising the pH to above the pKb through addition of base ortreatment with a basic ion exchange resin.

For compounds containing both amine groups and carboxylic acid groupssuch as but not limited to amino acids, these compounds may be formed orconverted to their ionic salt form by either 1) acid addition salts,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids such as acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-acid), carboxylic acid, glucoheptonicacid, 4,4′-methylenebis-(3-hydroxy-2-ene-l-carboxylic 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid. Acceptable inorganic bases includealuminum hydroxide, calcium hydroxide, potassium hydroxide, sodiumcarbonate, sodium hydroxide, and the like or 2) when an acidic protonpresent in the parent compound either is replaced by a metal ion, e.g.,an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base. Acceptable organic bases are known inthe art and include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases are known in the art and include aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like. The salt can be isolated as is from the system or converted tothe free acid by reducing the pH to below the pKa through addition ofacid or treatment with an acidic ion exchange resin.

It has been discovered that appropriate non-natural pathways,feedstocks, microorganisms, attenuation strategies to themicroorganism's biochemical network and cultivation strategies may becombined to efficiently produce 7-aminoheptanoate as a C7 (7-carbon)building block, or convert 7-aminoheptanoate to other C7 building blockssuch as pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or1,7-heptanediol or the corresponding salts thereof.

In some embodiments, a terminal carboxyl group can be enzymaticallyformed using a thioesterase, a CoA transferase, a ω-transaminase, analdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a6-oxohexanoate dehydrogenase or a 7-oxoheptanoate dehydrogenase. SeeFIG. 2 and FIG. 3.

In some embodiments, a terminal amine group can be enzymatically formedusing a carboxylate reductase, a ω-transaminase or a deacylase. See FIG.4. The ω-transaminase can have at least 70% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NOs. 7-12. Furthermore,the ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NOs. 7-12 and be capable oftransferring at least one amine group separated from a carbonyl group byat least one methylene insertion.

In some embodiments, a terminal hydroxyl group can be enzymaticallyformed using an alcohol dehydrogenase. See FIG. 5 and FIG. 6.

In one aspect, this document features a method of producingN-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof. The methodincludes enzymatically converting β-alanine toN-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof viaN-acetyl-3-aminopropanoate and N-acetyl-3-aminopropanoyl-CoA. β-alanineis converted to N-acetyl-3-aminopropanoate using a polypeptide havingthe activity of an acetyl-transferase classified under EC 2.3.1.-.N-acetyl-3-aminopropanoate is converted to N-acetyl-3-aminopropanoyl-CoAusing a polypeptide having the activity of a CoA ligase classified underEC 6.2.1.- or a CoA-transferase classified under EC 2.8.3.-.

N-acetyl-3-aminopropanoyl-CoA is converted toN-acetyl-5-amino-3-oxopentanoyl-CoA using a polypeptide having theactivity of a β-ketoacyl synthase classified under EC. 2.3.1.- (e.g., EC2.3.1.180) or a β-ketothiolase classified under EC. 2.3.1.- (e.g., EC2.3.1.16 or EC 2.3.1.174) The polypeptide having the activity of aβ-ketothiolase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:13. Furthermore, thepolypeptide having the activity of a β-ketothiolase can have at least70% sequence identity to the amino acid sequence set forth in SEQ IDNO:1 or SEQ ID NO:13 and be capable of convertingN-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA.The polypeptide having the activity of a β-ketoacyl synthase can have atleast 70% sequence identity to the amino acid sequence set forth in SEQID NO: 14. Furthermore, the polypeptide having the activity of aβ-ketoacyl synthase can have at least 70% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 14 and be capable of convertingN-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA.

The method can include enzymatically convertingN-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof to7-aminoheptanoate using a plurality of polypeptides having theactivities of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase,a trans-2-enoyl-CoA reductase, a β-ketothiolase, a thioesterase or a CoAtransferase and a deacetylase.

The polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenasecan be classified under EC 1.1.1.35, EC 1.1.1.36, EC 1.1.1.100 or EC1.1.1.157. The polypeptide having the activity of an enoyl-CoA hydratasecan be classified under EC 4.2.1.17 or EC 4.2.1.119. The polypeptidehaving the activity of a trans-2-enoyl-CoA reductase can be classifiedunder EC 1.3.1.38, EC 1.3.1.44 or EC 1.3.1.8. The polypeptide having theactivity of a β-ketothiolase can be classified under EC 2.3.1.16 or EC2.3.1.174. The polypeptide having the activity of a thioesterase or CoAtransferase can be classified under EC 3.1.2.- or EC 2.8.3.-respectively. The polypeptide having the activity of a deacetylase canbe classified under EC 3.5.1.-.

In one aspect, this document features a method for biosynthesizing7-aminoheptanoate or the salt thereof. The method includes enzymaticallyconverting N-acetyl-3-aminopropanoate toN-acetyl-5-amino-3-oxopentanoyl-CoA via N-acetyl-3-aminopropanoyl-CoA.N-acetyl-3-aminopropanoate is converted to N-acetyl-3-aminopropanoyl-CoAusing a polypeptide having the activity of a CoA ligase classified underEC 6.2.1.- or a CoA-transferase classified under EC 2.8.3.-.N-acetyl-3-aminopropanoyl-CoA is converted toN-acetyl-5-amino-3-oxopentanoyl-CoA using a polypeptide having theactivity of a β-ketoacyl synthase classified under EC 2.3.1.- (e.g., EC2.3.1.180) or a β-ketothiolase classified under EC. 2.3.1.- (e.g.,EC2.3.1.16 or EC 2.3.1.174). The β- ketothiolase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO:1 orSEQ ID NO:13. Furthermore, the β- ketothiolase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO:1 orSEQ ID NO:13 and be capable of converting N-acetyl-3-aminopropanoyl-CoAto N-acetyl-5-amino-3-oxopentanoyl-CoA. The β-ketoacyl synthase can haveat least 70% sequence identity to the amino acid sequence set forth inSEQ ID NO:14. Furthermore, the β-ketoacyl synthase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO:14and be capable of converting N-acetyl-3-aminopropanoyl-CoA toN-acetyl-5-amino-3-oxopentanoyl-CoA.

N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof can be convertedto N-acetyl-5-amino-3-hydroxypentanoyl-CoA using a polypeptide havingthe activity of a 3-hydroxyacyl-CoA dehydrogenase.N-acetyl-5-amino-3-hydroxypentanoyl-CoA can be converted toN-acetyl-5-amino-pent-2-enoyl-CoA using a polypeptide having theactivity of an enoyl-CoA hydratase. N-acetyl-5-amino-pent-2-enoyl-CoAcan be converted to N-acetyl-5-amino-pentanoyl-CoA using a polypeptidehaving the activity of a trans-2-enoyl-CoA reductase.N-acetyl-5-amino-pentanoyl-CoA can be converted toN-acetyl-7-amino-3-oxoheptanoyl-CoA using a polypeptide having theactivity of a β-ketothiolase. N-acetyl-7-amino-3-oxoheptanoyl-CoA can beconverted to N-acetyl-7-amino-3-hydroxyheptanoyl-CoA using a polypeptidehaving the activity of a 3-hydroxyacyl-CoA dehydrogenase.N-acetyl-7-amino-3-hydroxyheptanoyl-CoA can be converted toN-acetyl-7-amino-hept-2-enoyl-CoA using a polypeptide having theactivity of an enoyl-CoA hydratase. N-acetyl-7-amino-hept-2-enoyl-CoAcan be converted to N-acetyl-7-amino-heptanoyl-CoA using a polypeptidehaving the activity of a trans-2-enoyl-CoA reductase.N-acetyl-7-aminoheptanoyl-CoA can be converted toN-acetyl-7-amino-heptanoate using a polypeptide having the activity of athioesterase or a CoA transferase. N-acetyl-7-amino-heptanoate can beconverted to 7-aminoheptanoate using a polypeptide having the activityof a deacetylase.

Any of the methods further can include enzymatically converting7-aminoheptanoate to pimelic acid, 7-hydroxyheptanoate,heptamethylenediamine or 1,7-heptanediol or the corresponding saltsthereof in one or more steps.

For example, 7-aminoheptanoate can be enzymatically converted to pimelicacid using one or more polypeptides having the activity of aω-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, a 5-oxopentanoate dehydrogenase or an aldehydedehydrogenase. See FIG. 3.

For example, 7-aminoheptanoate and 7-hydroxyheptanoate can be convertedto heptamethylenediamine using one or more polypeptides having theactivity of a carboxylate reductase, a ω-transaminase, an alcoholdehydrogenase, an N-acetyltransferase, and a deacylase. See FIG. 4.

For example, 7-aminoheptanoate can be converted to 7-hydroxyheptanoateusing one or more polypeptides having the activity of an alcoholdehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutanoate dehydrogenase, or a ω-transaminase.See FIG. 5.

For example, 7-aminoheptanoate can be converted to 7-hydroxyheptanoate(see FIG. 5) and subsequently 7-hydroxyheptanoate can be converted to1,7-heptanediol using polypeptides having the activity of a carboxylatereductase and an alcohol dehydrogenase. See FIG. 6.

The ω-transaminase as described in any of the figures can have at least70% sequence identity to any one of the amino acid sequences set forthin SEQ ID NO. 7-12. Furthermore, the ω-transaminase as described in anyof the figures can have at least 70% sequence identity to any one of theamino acid sequences set forth in SEQ ID NO. 7-12 and be capable oftransferring at least one amine group separated from a carboxyl group byat least one methylene insertion.

The carboxylate reductase as described in any of the figures can have atleast 70% sequence identity to any one of the amino acid sequences setforth in SEQ D NO. 2-6 and 15. Furthermore, the carboxylate reductase asdescribed in any of the figures can have at least 70% sequence identityto any one of the amino acid sequences set forth in SEQ D NO. 2-6 and 15and be capable of reducing a carboxyl group to a terminal aldehyde.

In any of the methods, N-acetyl-3-aminopropanoate can be enzymaticallyproduced from β-alanine, which itself can be enzymatically produced frommalonyl-CoA using polypeptides having the activity of a malonyl-CoAreductase and a β-alanine-pyruvate aminotransferase or from L-aspartateusing a polypeptide having the activity of an aspartate α-decarboxylase.

In any of the methods described herein, pimelic acid can be produced byforming the second terminal functional group in pimelate semialdehyde(also known as 7-oxoheptanoate) using a polypeptide having the activityof (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, or (ii) a5-oxopentanoate dehydrogenase classified under EC 1.2.1.- such asencoded by CpnE, a 6-oxohexanoate dehydrogenase classified under EC1.2.1.63 such as that encoded by ChnE or a 7-oxoheptanoate dehydrogenaseclassified under EC 1.2.1.- (e.g., the gene product of ThnG). See FIG.3.

In any of the methods described herein, 7-hydroxyheptanoic acid can beproduced by forming the second terminal functional group in pimelatesemialdehyde using a polypeptide having the activity of an alcoholdehydrogenase classified under EC 1.1.1.-, 6-hydroxyhexanoatedehydrogenase classified under EC 1.1.1.258 such as the gene product ofChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162);a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.- such asthe gene product of cpnD, or 4-hydroxybutanoate dehydrogenase classifiedunder EC 1.1.1.61 such as the gene product of gabD. See FIG. 5.

In any of the methods described herein, heptamethylenediamine can beproduced by forming a second terminal functional group in (i)7-aminoheptanal using a polypeptide having the activity of aω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29,EC 2.6.1.48 or EC 2.6.1.82 or in (ii) N7-acetyl-1,7-diaminoheptane usinga deacylase classified, for example, under EC 3.5.1.62. See FIG. 4.

In any of the methods described herein, 1,7-heptanediol can be producedby forming the second terminal functional group in 7-hydroxyheptanalusing a polypeptide having the activity of an alcohol dehydrogenaseclassified under EC 1.1.1.- (e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or1.1.1.184) such as that encoded by YMR318C, YqhD or CAA81612.1. See FIG.6.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcycloheptane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

In some embodiments, the microorganism's tolerance to highconcentrations of one or more C7 (7-carbon) building blocks is improvedthrough continuous cultivation in a selective environment.

In some embodiments, the microorganism's biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofacetyl-CoA and β-alanine, (2) create an NADH or NADPH imbalance that mayonly be balanced via the formation of one or more C7 building blocks,(3) prevent degradation of central metabolites, central precursorsleading to and including C7 building blocks and (4) ensure efficientefflux from the cell.

In some embodiments, a cultivation strategy is used to achieveanaerobic, micro-aerobic, or aerobic cultivation conditions.

In some embodiments, the cultivation strategy includes limitingnutrients, such as limiting nitrogen, phosphate or oxygen.

In some embodiments, one or more C7 building blocks are produced by asingle type of microorganism, e.g., a recombinant microorganismcontaining one or more exogenous nucleic acids, using, for example, afermentation strategy. In some embodiments, one or more C7 buildingblocks are produced by a single type of microorganism having one or moreexogenous nucleic acids which encode a polypeptide having an activity of3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, atrans-2-enoyl-CoA reductase, a β-ketothiolase, a β-ketoacyl synthase, athioesterase or a CoA transferase and a deacetylase, using, for example,a fermentation strategy. In another aspect, this document features arecombinant microorganism that includes at least one exogenous nucleicacid encoding a polypeptide having the activity of (i) a β-ketoacylsynthase, (ii) a β-ketothiolase, (iii) a thioesterase or a CoAtransferase, (iv) a deacetylase and one or more of (v) a3-hydroxyacyl-CoA dehydrogenase, (vi) an enoyl-CoA hydratase, and (vii)a trans-2-enoyl-CoA reductase, the microorganism producing7-aminoheptanoate or a corresponding salt thereof. See FIG. 1 and FIG.2.

A microorganism producing 7-aminoheptanoate further can include one ormore of the following exogenous polypeptides having the activity of: aω-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, a 5-oxopentanoate dehydrogenase, or an aldehydedehydrogenase, the microorganism further producing pimelic acid. SeeFIG. 3.

A microorganism producing 7-aminoheptanoate or 7-hydroxyheptanoatefurther can include one or more of the following exogenous polypeptideshaving the activity of: a carboxylate reductase, a ω-transaminase, adeacylase, an N-acetyl transferase, or an alcohol dehydrogenase, saidmicroorganism further producing heptamethylenediamine. See FIG. 4.

A microorganism producing 7-aminoheptanoate further can include one ormore of the following exogenous polypeptides having the activity of: aω-transaminase, a 6-hydroxyhexanoate dehydrogenase, a 4-hydroxybutanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, and an alcoholdehydrogenase, the microorganism further producing 7-hydroxyheptanoate.See FIG. 5.

A microorganism producing 7-hydroxyheptanoate further can include anexogenous polypeptide having the activity of a carboxylate reductase andan exogenous polypeptide having the activity of an alcoholdehydrogenase, the microorganism further producing 1,7-heptanediol. SeeFIG. 6.

Any of the recombinant microorganisms described herein further caninclude one or more of the following exogenous polypeptides having theactivity of: an aspartate-α-decarboxylase; a malonyl-CoA reductase; aβ-alanine-pyruvate aminotransferase; an N-acetyl transferase; athioesterase; a CoA-transferase; and a deactylase.

Any of the recombinant microorganisms can be a prokaryote such as aprokaryote from a genus selected from the group consisting ofEscherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas;Delftia; Bacilluss; Lactobacillus; Lactococcus; and Rhodococcus. Forexample, the prokaryote can be selected from the group consisting ofEscherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum,Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator,Cupriavidus metallidurans. Pseudomonas fluorescens, Pseudomonas putida,Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis,Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi.Such prokaryotes also can be sources of genes for constructingrecombinant cells described herein that are capable of producing C7building blocks.

Any of the recombinant microorganisms can be a eukaryote such as aeukaryote from a genus selected from the group consisting ofAspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia,Debaryomyces, Arxula, and Kluyveromyces. For example, the eukaryote canbe selected from the group consisting of Aspergillus niger,Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica,Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans,and Kluyveromyces lactis. Such eukaryotes also can be sources of genesfor constructing recombinant cells described herein that are capable ofproducing C7 building blocks.

Any of the recombinant microorganisms described herein further caninclude attenuation of one or more of the following enzymes: apolyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, an alcoholdehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvatedecarboxylase, a glucose-6-phosphate isomerase, NADH-consumingtranshydrogenase, an NADH-specific glutamate dehydrogenase, aNADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoAdehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocksand central precursors as substrates; a butyryl-CoA dehydrogenase; or anadipyl-CoA synthetase.

Any of the recombinant microorganisms described herein further canoverexpress one or more genes encoding: an acetyl-CoA synthetase, a6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotidetranshydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; aglucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamatedehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; adiamine transporter; a dicarboxylate transporter; and/or a multidrugtransporter.

In another aspect of the invention, this document features anon-naturally occurring microorganism comprising at least one exogenousnucleic acid encoding at least one polypeptide having the activity of atleast one enzyme, at least one substrate and at least one product,depicted in any one of FIGS. 1 to 6.

In another aspect of the invention, this document features a pluralityof nucleic acid constructs or expression vectors comprising apolynucleotide encoding a polypeptide having enzymatic activitiescorresponding to the polypeptides as set out in SEQ ID NO:1 to SEQ IDNO: 15 and to polypeptides having at least 70% sequence identity to thepolypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15. (See FIG. 7).

In another aspect of the invention, this document features a compositioncomprising a nucleic acid construct or expression vector comprising apolynucleotide encoding a polypeptide having enzymatic activitiescorresponding to the polypeptides as set out in SEQ ID NO:1 to SEQ IDNO: 15 and to polypeptides having at least 70% sequence identity to thepolypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15. (See FIG. 7).

In another aspect of the invention, this document features a culturemedium comprising a nucleic acid construct or expression vectorcomprising a polynucleotide encoding a polypeptide having enzymaticactivities corresponding to the polypeptides as set out in SEQ ID NO:1to SEQ ID NO: 15 and to polypeptides having at least 70% sequenceidentity to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15.(See FIG. 7).

In another aspect of the invention, this document features anon-naturally occurring biochemical network comprisingN-acetyl-3-aminopropanoyl-CoA, an exogenous nucleic acid encoding apolypeptide having the activity of a β-ketothiolase or a β-ketoacylsynthase classified under EC. 2.3.1, and anN-acetyl-5-amino-3-oxopentanoyl-CoA.

In another aspect of the invention, this document features anon-naturally occurring biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having the enzymaticactivity of (i) a β-ketoacyl synthase and/or a β-ketothiolase, (ii) athioesterase or a CoA transferase, (iii) a deacetylase, and one or moreof (iv) 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase,and (v) a trans-2-enoyl-CoA reductase, said microorganism producing7-aminoheptanoate.

In another aspect of the invention, this document features means forproducing 7-aminoheptanoate, comprising culturing a non-naturallyoccurring microorganism comprising at least one exogenous nucleic acidencoding a polypeptide having the enzymatic activity of (i) a β-ketoacylsynthase and/or a β-ketothiolase, (ii) a thioesterase or a CoAtransferase, (iii) a deacetylase, and one or more of (iv) a3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) atrans-2-enoyl-CoA reductase expressed in a sufficient amount in saidmicroorganism to produce 7-amino-heptanoate.

In another aspect of the invention, this document features abio-derived, bio-based or fermentation-derived product, wherein saidproduct comprises: i. a composition comprising at least one bio-derived,bio-based or fermentation-derived compound according to any one ofclaims 15-28 or any combination thereof, ii. a bio-derived, bio-based orfermentation-derived polymer comprising the bio-derived, bio-based orfermentation-derived composition or compound of i., or any combinationthereof, iii. a bio-derived, bio-based or fermentation-derived resincomprising the bio-derived, bio-based or fermentation-derived compoundor bio-derived, bio-based or fermentation-derived composition of i. orany combination thereof or the bio-derived, bio-based orfermentation-derived polymer of ii. or any combination thereof, iv. amolded substance obtained by molding the bio-derived, bio-based orfermentation-derived polymer of ii. or the bio-derived, bio-based orfermentation-derived resin of iii., or any combination thereof, v. abio-derived, bio-based or fermentation-derived formulation comprisingthe bio-derived, bio-based or fermentation-derived composition of i.,bio-derived, bio-based or fermentation-derived compound of i.,bio-derived, bio-based or fermentation-derived polymer of ii.,bio-derived, bio-based or fermentation-derived resin of iii., orbio-derived, bio-based or fermentation-derived molded substance of iv,or any combination thereof, or vi. a bio-derived, bio-based orfermentation-derived semi-solid or a non-semi-solid stream, comprisingthe bio-derived, bio-based or fermentation-derived composition of i.,bio-derived, bio-based or fermentation-derived compound of i.,bio-derived, bio-based or fermentation-derived polymer of ii.,bio-derived, bio-based or fermentation-derived resin of iii.,bio-derived, bio-based or fermentation-derived formulation of v., orbio-derived, bio-based or fermentation-derived molded substance of iv.,or any combination thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from theapplication, including the written description and drawings, and theclaims. The word “comprising” in the claims may be replaced by“consisting essentially of” or with “consisting of,” according tostandard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of exemplary biochemical pathways leading toN-acetyl-7-aminoheptanoyl-CoA using malonyl-CoA or L-aspartate ascentral metabolites.

FIG. 2 is a schematic of an exemplary biochemical pathway leading to7-aminoheptanoate using N-acetyl-7-aminoheptanoyl-CoA as a precursor.

FIG. 3 is a schematic of exemplary biochemical pathways leading topimelic acid using 7-aminoheptanoate as a central precursor.

FIG. 4 is a schematic of exemplary biochemical pathways leading toheptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate,pimelate semialdehyde or 1,7-heptanediol as a central precursor.

FIG. 5 is a schematic of exemplary biochemical pathways leading to7-hydroxyheptanoate using 7-aminoheptanoate as a central precursor.

FIG. 6 is a schematic of an exemplary biochemical pathway leading to1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.

FIG. 7 contains the amino acid sequences of a Cupriavidus necatorβ-ketothiolase (see GenBank Accession No. AAC38322.1, SEQ ID NO: 1), aMycobacterium marinum carboxylate reductase (see Genbank Accession No.ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis carboxylatereductase (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aSegniliparus rugosus carboxylate reductase (see Genbank Accession No.EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliensecarboxylatereductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 5), aSegniliparus rotundus carboxylate reductase (see Genbank Accession No.ADG98140.1, SEQ ID NO: 6), a Chromobacterium violaceum ω-transaminase(see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa ω-transaminase (see Genbank Accession No. AAG08191.1, SEQ IDNO: 8), a Pseudomonas syringae ω-transaminase (see Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides ω-transaminase (seeGenbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coliω-transaminase (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11), aVibrio fluvialis ω-transaminase (See Genbank Accession No. AEA39183.1,SEQ ID NO: 1 CAA74523.12), an Escherichia coli β-ketothiolase (seeGenBank Accession No. AAC74479.1, SEQ ID NO: 13), a Bacillus subtilisβ-ketoacyl synthase (see GenBank Accession No. CAA74523.1, SEQ ID NO:14), a Mycobacterium smegmatis carboxylate reductase (see GenBankAccession No. ABK75684.1, SEQ ID NO: 15), a Cupriavidus necatorbeta-ketothiolase (see GenBank Accession No. AAC38322.1, SEQ ID NO: 16),an Escherichia coli (see Genbank Accession No. AAC74479.1, SEQ ID NO:17), a Clostridium propionicum acetate/propionate CoA transferase (seeGenbank Accession No. CAB77207.1, SEQ ID NO: 18), a Clostridiumaminobutyricum 4-hydroxybutyrate-CoA transferase (see Genbank AccessionNo. CAB60036.2, SEQ ID NO: 19), a Citrobacter sp. Al acetyl-CoAhydrolase/transferase transferase (see Genbank Accession No. EJF23789.1,SEQ ID NO: 20), and an Acetobacter aceti succinyl-CoA:acetateCoA-transferase (see Genbank Accesssion No. ACD85596.1, SEQ ID NO: 21).

FIG. 8 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of six carboxylate reductase preparations in enzyme onlycontrols (no substrate).

FIG. 9 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof two carboxylate reductase preparations for converting pimelate topimelate semialdehyde relative to the empty vector control.

FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof six carboxylate reductase preparations for converting7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty vectorcontrol.

FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof three carboxylate reductase preparations for convertingN7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal relative to theempty vector control.

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofa carboxylate reductase preparation for converting pimelate semialdehydeto heptanedial relative to the empty vector control.

FIG. 13 is a bar graph summarizing the percent conversion of pyruvate toL-alanine (mol/mol) as a measure of the ω-transaminase activity of theenzyme only controls (no substrate).

FIG. 14 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of four ω-transaminase preparations for converting7-aminoheptanoate to pimelate semialdehyde relative to the empty vectorcontrol.

FIG. 15 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity of three ω-transaminase preparations for converting pimelatesemialdehyde to 7-aminoheptanoate relative to the empty vector control.

FIG. 16 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of six ω-transaminase preparations for convertingheptamethylenediamine to 7-aminoheptanal relative to the empty vectorcontrol.

FIG. 17 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of six ω-transaminase preparations for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal relative tothe empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of three ω-transaminase preparations for converting7-aminoheptanol to 7-oxoheptanol relative to the empty vector control.

FIG. 19 is a schematic of the exemplary enzymatic reactions performedwith 4-hydroxybutyrate-CoA transferase using either N-acetyl-β-alanine(AC5) or 5-ethanamidopentanoic acid (AC7) as substrates for theformation of 5-ethanamido-3-oxopentanoyl-CoA and7-ethanamido-3-oxoheptanoyl-CoA, respectively.

FIG. 20 is a LC-MS chromatogram of distinct peaks of chemical abundanceseparated by retention times as a measure of enzyme activity for of4-hydroxybutyrate-CoA transferase for converting 5-ethanamidopentanoicacid into products, 5-ethanamidopentanoyl-CoA (g) and7-ethanamido-3-oxopentanoyl-CoA (h).

FIG. 21 is a LC-MS ESI mass spectrum performed in positive mode thatidentifies the product of peak (g) (see chromatogram of FIG. 20) asethanamidopentanoyl-CoA by comparison of the observed and expectedmasses for the [M+H]⁺ and [M+2H]²⁺ species. Expected [M+H]⁺ for products(g): 909.2017 (1 charge) & [M+2H]²⁺: 445.1044 (2 charges).

FIG. 22 is a LC-MS chromatogram of distinct peaks of chemical abundanceseparated by retention times as a measure of enzyme activity for of4-hydroxybutyrate-CoA transferase for converting5-ethanamidopentanoyl-CoA into the product.7-ethanamido-3-oxopentanoyl-CoA (h).

FIG. 23 is a LC-MS ESI mass spectrum performed in positive mode thatidentifies the product of peak (h) (see chromatogram of FIG. 22) asethanamido-3-oxopentanoyl-CoA by comparison of the observed and expectedmasses for the [M+H]⁺ and [M+2H]²⁺ species. Expected [M+H]⁺ for products(h): 951.2120 (1 charge) & [M+2H]2+: 476.1097 (2 charges).

DETAILED DESCRIPTION

In general, this document provides enzymes, non-natural pathways,cultivation strategies, feedstocks, microorganisms and attenuations tothe microorganism's biochemical network, for producing 7-aminoheptanoateor one or more of pimelic acid, 7-hydroxyheptanoic acid,heptamethylenediamine or 1,7-heptanediol or the corresponding saltsthereof, all of which are referred to as C7 building blocks herein. Theterm “C7 building block” is used to denote a seven (7) carbon chainaliphatic backbone. As used herein, the term “central precursor” is usedto denote any metabolite in any metabolic pathway shown herein leadingto the synthesis of a C7 building block. The term “central metabolite”is used herein to denote a metabolite that is produced in allmicroorganisms to support growth.

Microorganisms described herein can include endogenous pathways that canbe manipulated such that 7-aminoheptanoate or one or more other C7building blocks can be produced. In an endogenous pathway, themicroorganism naturally expresses all of the enzymes catalyzing thereactions within the pathway. A microorganism containing an engineeredpathway does not naturally express all of the enzymes catalyzing thereactions within the pathway but has been engineered such that all ofthe enzymes within the pathway are expressed in the microorganism.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a microorganism refers to a nucleic acid that does notoccur in (and cannot be obtained from) a cell of that particular type asit is found in nature or a protein encoded by such a nucleic acid. Thus,a non-naturally-occurring nucleic acid is considered to be exogenous toa microorganism once in the microorganism. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a microorganism once introduced into themicroorganism, since that nucleic acid molecule as a whole (genomic DNAplus vector DNA) does not exist in nature. Thus, any vector,autonomously replicating plasmid, or virus (e.g., retrovirus,adenovirus, or herpes virus) that as a whole does not exist in nature isconsidered to be non-naturally-occurring nucleic acid. It follows thatgenomic DNA fragments produced by PCR or restriction endonucleasetreatment as well as cDNAs are considered to be non-naturally-occurringnucleic acid since they exist as separate molecules not found in nature.It also follows that any nucleic acid containing a promoter sequence andpolypeptide-encoding sequence (e.g., cDNA or genomic DNA) in anarrangement not found in nature is non-naturally-occurring nucleic acid.A nucleic acid that is naturally-occurring can be exogenous to aparticular microorganism. For example, an entire chromosome isolatedfrom a cell of yeast x is an exogenous nucleic acid with respect to acell of yeast y once that chromosome is introduced into a cell of yeasty.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a microorganism refers toa nucleic acid (or protein) that does occur in (and can be obtainedfrom) that particular microorganism as it is found in nature. Moreover,a cell “endogenously expressing” a nucleic acid (or protein) expressesthat nucleic acid (or protein) as does a microorganism of the sameparticular type as it is found in nature. Moreover, a microorganism“endogenously producing” or that “endogenously produces” a nucleic acid,protein, or other compound produces that nucleic acid, protein, orcompound as does a microorganism of the same particular type as it isfound in nature.

For example, depending on the microorganism and the compounds producedby the microorganism, one or more of the following polypeptides havingthe following specific enzymatic activities may be expressed in themicroorganism in addition to a β-ketoacyl synthase and/or aβ-ketothiolase:a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoAhydratase, a trans-2-enoyl-CoA reductase, a thioesterase, a CoAtransferase, a deacetylase, an aldehyde dehydrogenase, an alcoholdehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-hydroxyhexanoatedehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a carboxylate reductase, an N-acetyl transferase, or aω-transaminase. In recombinant microorganisms expressing a polypeptidehaving the activity of a carboxylate reductase, a phosphopantetheinyltransferase also can be expressed as it enhances activity of thecarboxylate reductase.

For example, a recombinant microorganism can include a polypeptidehaving the activity of an exogenous β-ketoacyl synthase or aβ-ketothiolase and produce N-acetyl-5-amino-3-oxopentanoyl-CoA or a saltthereof from N-acetyl-3-aminopropanoyl-CoA. TheN-acetyl-5-amino-3-oxopentanoyl-CoA or salt thereof can be convertedenzymatically to N-acetyl-7-aminoheptanoyl-CoA and subsequently to7-aminoheptanoate.

For example, a recombinant microorganism can include a polypeptidehaving the activity of an exogenous β-ketoacyl synthase and aβ-ketothiolase, an exogenous thioesterase or CoA-transferase, adeacetylase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase,and a trans-2-enoyl-CoA reductase and produce 7-aminoheptanoate.

For example, a recombinant microorganism producing 7-aminoheptanoate caninclude one or more of the following exogenous polypeptides having theenzymatic activity of: a ω-transaminase, a 7-oxoheptanoatedehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, or an aldehyde dehydrogenase, and further produce pimelicacid. For example, a recombinant microorganism producing7-aminoheptanoate can include an exogenous ω-transaminase and analdehyde dehydrogenase and produce pimelic acid. For example, arecombinant microorganism producing 7-aminoheptanoate can include anexogenous polypeptide having the activity of a ω-transaminase and one ofthe following exogenous polypeptides having the enzymatic activity of: a5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a7-oxoheptanoate dehydrogenase, and produce pimelic acid.

For example, a recombinant microorganism producing 7-aminoheptanoate caninclude one or more of the following exogenous polypeptides having theenzymatic activity of: a ω-transaminase, an alcohol dehydrogenase, a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase,4-hydroxybutanoate dehydrogenase, and further produce7-hydroxyheptanoate. For example, a recombinant microorganism producing7-aminoheptanoate can include an exogenous polypeptide having theactivity of an alcohol dehydrogenase and an exogenous polypeptide havingthe activity of a ω-transaminase and produce 7-hydroxyheptanoate. Forexample, a recombinant microorganism producing 7-aminoheptanoate caninclude an exogenous polypeptide having the activity of a6-hydroxyhexanoate dehydrogenase and an exogenous polypeptide having theactivity of a ω-transaminase and produce 7-hydroxyheptanoate. Forexample, a recombinant microorganism producing 7-aminoheptanoate caninclude an exogenous polypeptide having the activity of a5-hydroxypentanoate dehydrogenase and an exogenous polypeptide havingthe activity of a ω-transaminase and produce 7-hydroxyheptanoate. Forexample, a recombinant microorganism producing 7-aminoheptanoate caninclude an exogenous polypeptide having the activity of a4-hydroxybutanoate dehydrogenase and an exogenous polypeptide having theactivity of a ω-transaminase and produce 7-hydroxyheptanoate.

For example, a recombinant microorganism producing 7-aminoheptanoate caninclude one or more of the following exogenous polypeptides having theactivity of: a carboxylate reductase, a ω-transaminase, a deacetylase,an N-acetyl transferase or an alcohol dehydrogenase, and produceheptamethylenediamine. For example, a recombinant microorganismproducing 7-aminoheptanoate can include an exogenous polypeptide havingthe activity of a carboxylate reductase and one or more exogenouspolypeptides having the activity of transaminases (e.g., oneω-transaminase or two different transaminases) and produceheptamethylenediamine. For example, a recombinant microorganismproducing 7-aminoheptanoate can include an exogenous polypeptide havingthe activity of a carboxylate reductase, an exogenous polypeptide havingthe activity of a alcohol dehydrogenase, and one or more exogenouspolypeptides having the activity of transaminases (e.g., oneω-transaminase or two different transaminases), and produceheptamethylenediamine. For example, a recombinant microorganismproducing 7-aminoheptanoate can include an exogenous polypeptide havingthe activity of an N-acetyl transferase, a carboxylate reductase, adeacylase, and one or more exogenous transaminases (e.g., oneω-transaminase or two different transaminases) and produceheptamethylenediamine. For example, a recombinant microorganismproducing7-aminoheptanoate can include one or more exogenous polypeptide havingthe activity of an alcohol dehydrogenase, and one or more exogenouspolypeptides having the activity of transaminases (e.g., oneω-transaminase, or two or three different transaminases) and produceheptamethylenediamine.

For example, a recombinant microorganism producing 7-hydroxyheptanoatecan include the following exogenous polypeptides having the enzymaticactivity of: a carboxylate reductase and an exogenous alcoholdehydrogenase, and further produce 1,7-heptanediol.

In any of the recombinant microorganisms, the recombinant microorganismalso can include one or more (e.g., one, two or three) of the followingexogenous enzymes used to convert either malonyl-CoA or L-aspartate toβ-alanine: a malonyl-CoA reductase, an aspartate α-decarboxylase and aβ-alanine-pyruvate aminotransferase. For example, a recombinantmicroorganism can include an exogenous malonyl-CoA reductase and aβ-alanine-pyruvate aminotransferase and produce β-alanine. For example,a recombinant microorganism can include an exogenous aspartateα-decarboxylase and produce β-alanine.

In any of the recombinant microorganisms, the recombinant microorganismalso can include following the exogenous enzyme used to convertβ-alanine to N-acetyl-3-aminopropanoate: an N-acetyl-transferase.

In any of the recombinant microorganisms, the recombinant microorganismalso can include one or more (e.g., one or two) of the followingexogenous enzymes used to convert N-acetyl-3-aminopropanoate toN-acetyl-3-aminopropanoyl-CoA: a CoA transferase or a CoA ligase.

In any of the recombinant microorganisms, the recombinant microorganismalso can include one or more (e.g., one or two) of the followingexogenous enzymes used to convert 5-ethanamidopentanoic acid to5-ethanamidopentanoyl-CoA: a CoA transferase or a CoA ligase.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genera, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank or EMBL.

Any of the enzymes described herein that can be used for production ofone or more C7 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of thecorresponding wild-type enzyme. It will be appreciated that the sequenceidentity can be determined on the basis of the mature enzyme (e.g., withany signal sequence removed) or on the basis of the immature enzyme(e.g., with any signal sequence included). It also will be appreciatedthat the initial methionine residue may or may not be present on any ofthe enzyme sequences described herein.

For example, a β-ketothiolase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acidsequence of a Cupriavidus necator β-ketothiolase (see GenBank AccessionNo. AAC38322.1, SEQ ID NO: 1) or an Escherichia coli β-ketothiolase (seeGenBank Accession No. AAC74479.1, SEQ ID NO: 13) See FIG. 7.

For example, a β-ketoacyl synthase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acidsequence of a Bacillus subtilis β-ketoacyl synthase (see GenBankAccession No. CAA74523.1, SEQ ID NO: 14). See FIG. 7.

For example, a CoA-transferase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acidsequence of a Clostridium aminobutyrium (see GenBank Accession No.CAB60036.2, SEQ ID NO: 19). See FIG. 7.

For example, a carboxylate reductase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acidsequence of a Mycobacterium marinum carboxylate reductase (see GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (see Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliense carboxylatereductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 5), aSegniliparus rotundus carboxylate reductase (see Genbank Accession No.ADG98140.1, SEQ ID NO: 6) carboxylate reductase or a Mycobacteriumsmegmatis carboxylate reductase (see Genbank Accession No. ABK75684.1,SEQ ID NO: 15). See, FIG. 7.

For example, a ω-transaminase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acidsequence of a Chromobacterium violaceum ω-transaminase (see GenbankAccession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosaω-transaminase (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae ω-transaminase (see Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides ω-transaminase (seeGenbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia colitransaminase (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11), or aVibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 12)ω-transaminase. Some of these ω-transaminases are diamineω-transaminases. See, FIG. 7.

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (B12seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. B12seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seql.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\B12seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 100 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, 50 or 100) amino acid substitutions (e.g.,conservative substitutions). This applies to any of the enzymesdescribed herein and functional fragments. A conservative substitutionis a substitution of one amino acid for another with similarcharacteristics. Conservative substitutions include substitutions withinthe following groups: valine, alanine and glycine; leucine, valine, andisoleucine; aspartic acid and glutamic acid; asparagine and glutamine;serine, cysteine, and threonine; lysine and arginine; and phenylalanineand tyrosine. The nonpolar hydrophobic amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Any substitution of one member of the above-mentionedpolar, basic or acidic groups by another member of the same group can bedeemed a conservative substitution. By contrast, a nonconservativesubstitution is a substitution of one amino acid for another withdissimilar characteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 50 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., heptahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain microorganisms (e.g., yeast cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered microorganisms can naturally express none or some (e.g., oneor more, two or more, three or more, four or more, five or more, or sixor more) of the enzymes of the pathways described herein. Thus, apathway within an engineered microorganism can include all exogenousenzymes, or can include both endogenous and exogenous enzymes.Endogenous genes of the engineered microorganisms also can be disruptedto prevent the formation of undesirable metabolites or prevent the lossof intermediates in the pathway through other enzymes acting on suchintermediates. Engineered microorganisms can be referred to asrecombinant microorganisms or recombinant cells. As described hereinrecombinant microorganisms can include nucleic acids encoding one ormore of a β-ketoacyl synthase, a β-ketothiolase, a dehydrogenase, areductase, a hydratase, a CoA-transferase, a CoA-ligase, a thioesterase,a deacetylase, an N-acetyltransferase and ω-transaminase as describedherein.

In addition, the production of C7 building blocks can be performed invitro using the isolated enzymes described herein, using a lysate (e.g.,a cell lysate) from a microorganism as a source of the enzymes, or usinga plurality of lysates from different microorganisms as the source ofthe enzymes.

The reactions of the pathways described herein can be performed in oneor more microorganisms (a) naturally expressing one or more relevantenzymes, (b) genetically engineered to express one or more relevantenzymes, or (c) naturally expressing one or more relevant enzymes andgenetically engineered to express one or more relevant enzymes.Alternatively, relevant enzymes can be isolated, purified or extractedfrom of the above types of microorganism cells and used in a purified orsemi-purified form. Moreover, such extracts include lysates (e.g. celllysates) that can be used as sources of relevant enzymes. In the methodsprovided by the document, all the steps can be performed inmicroorganism cells, all the steps can be performed using extractedenzymes, or some of the steps can be performed in cells and others canbe performed using extracted enzymes.

Enzymes Enzymes generating N-acetyl-7-amino-heptanoyl-CoA

As depicted in FIG. 1, N-acetyl-7-amino-heptanoyl-CoA or a salt thereofcan be biosynthesized from malonyl-CoA or L-aspartate through theintermediate N-acetyl-5-amino-3-oxopentanoyl-CoA, which can be producedfrom N-acetyl-3-aminopropanoyl-CoA using a polypeptide having theactivity of a β-ketoacyl synthase or a β-ketothiolase. In someembodiments, a β-ketothiolase may be classified under EC 2.3.1.16, suchas the gene product of bktB or under EC 2.3.1.174 such as the geneproduct of paaJ. In some embodiments, a β-ketoacyl synthase may beclassified under EC 2.3.1.180 such as the gene product of fabH, under EC2.3.1.179 such as the gene product of fabF or under EC 2.3.1.41 such asthe gene product of fabB.

N-acetyl-3-aminopropanoyl-CoA or a salt thereof can be enzymaticallyconverted from N-acetyl-3-aminopropanoate using a polypeptide having theactivity of a CoA transferase classified, for example, under EC 2.8.3-or a CoA ligase classified, for example, under EC 6.2.1-.N-acetyl-3-aminopropanoate can be enzymatically produced from β-alanineusing a polypeptide having the activity of an N-acetyl transferaseclassified, for example, under EC 2.3.1.-, such as EC 2.3.1.13, EC2.3.1.17 or EC 2.3.1.32.

β-alanine itself can be enzymatically produced from malonyl-CoA usingpolypeptides having the activity of a malonyl-CoA-reductase and aβ-alanine-pyruvate aminotransferase or from L-aspartate using apolypeptide having the activity of an α-aspartate decarboxylase. In someembodiments, a malonyl-CoA-reductase may be classified under EC 1.2.1.75and a β-alanine-pyruvate aminotransferase may be classified under EC2.6.1.18. In some embodiments, an α-aspartate decarboxylase may beclassified under EC 4.1.1.11.

The intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA or salt thereof canbe converted to N-acetyl-7-amino-heptanoyl-CoA using polypeptides havingthe activity of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoAhydratase, a trans-2-enoyl-CoA reductase and a β-ketothiolase. In someembodiments, a 3-hydroxyacyl-CoA dehydrogenase may be classified, forexample, under EC 1.1.1.- such as EC 1.1.1.35 (e.g., the gene product offadB), EC 1.1.1.36 (e.g., the gene product of phaB), or EC 1.1.1.157(e.g., the gene product of hbd). In some embodiments, an enoyl-CoAhydratase may be classified under EC 4.2.1.17 such as the gene productof crt or under EC 4.2.1.119 such as the gene product of phaJ. In someembodiments, a trans-2-enoyl-CoA reductase may be classified, forexample, under EC 1.3.1.38 or EC 1.3.1.44, such as the gene product ofter (Nishimaki et al., J. Biochem., 1984, 95:1315-1321; Shen et al.,2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012,51:6827-6837) or EC 1.3.1.8 (Inui et al., Eur. J. Biochem., 1984, 142,121-126). In some embodiments, a β-ketothiolase may be classified underEC 2.3.1.16 such as the gene product of bktB or under EC 2.3.1.174 suchas the gene product of paaJ.

Enzymes generating 7-aminoheptanoate

As depicted in FIG. 2, N-acetyl-7-amino-heptanoyl-CoA is converted to7-aminoheptanoate using polypeptides having the activity of athioesterase or CoA-transferase and a deacetylase.

In some embodiments, a thioesterase may be classified under EC 3.1.2.-,resulting in the production of N-acetyl-7-aminoheptanoate. Thethioesterase can be the gene product of YciA or Acot13 (Cantu et al.,Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008,47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991,266(17):11044-11050). In some embodiments, a CoA-transferase may beclassified under, for example, EC 2.8.3.- such as the gene product ofcat2 from Clostridium kluyveri, abfT from Clostridium aminobutyricum orthe 4-hydroxybutyrate CoA-transferase from Clostridium viride.

In some embodiments, the first terminal amine group leading to thesynthesis of 7-aminoheptanoate is enzymatically formed by a deacetylaseclassified, for example, under EC 3.5.1.17 such as an acyl-lysinedeacetylase from Achromobacter pestifer (see, for example, Chibate etal., 1970, Methods Enzymol., 19:756-762).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis ofPimelic Acid

As depicted in FIG. 3, 7-aminoheptanoate can be enzymatically convertedto pimelic acid. The terminal carboxyl group leading to the productionof pimelic acid can be enzymatically formed using polypeptides havingthe activity of an aldehyde dehydrogenase, a 5-oxopentanoatedehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoatedehydrogenase.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid can be enzymatically formed in pimelatesemialdehyde by an aldehyde dehydrogenase classified under EC 1.2.1.3(Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192). See,FIG. 3.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed in pimelatesemialdehyde by EC 1.2.1.-such as a 5-oxopentanoate dehydrogenaseclassified, for example, under EC 1.2.1.20, such as the gene product ofCpnE, a 6-oxohexanoate dehydrogenase classified, for example, EC1.2.1.63 such as the gene product of ChnE from Acinetobacter sp., or a7-oxoheptanoate dehydrogenase such as the gene product of ThnG fromSphingomonas macrogolitabida (Iwaki et al., Appl. Environ. Microbiol.,1999, 65(11), 5158-5162; López-Sánchez et al., Appl. Environ.Microbiol., 2010, 76(1), 110-118)). See, FIG. 3.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis ofHeptamethylenediamine

As depicted in FIG. 4, terminal amine groups can be enzymatically formedor removed using polypeptides having the activity of a ω-transaminase ora deacetylase.

In some embodiments, a terminal amine group leading to the synthesis of7-aminoheptanoic acid is enzymatically formed in 7-aminoheptanal by aω-transaminase classified, for example, under EC 2.6.1.-, e.g., EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asthat obtained from Chromobacterium violaceum (Genbank Accession No.AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (Genbank Accession No.AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (Genbank Accession No.AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (Genbank AccessionNo. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (Genbank Accession No.AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride.See FIG. 7.

An additional ω-transaminase that can be used in the methods andmicroorganisms described herein is from Escherichia coli (GenbankAccession No. AAA57874.1, SEQ ID NO: 11). Some of the ω-transaminasesclassified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamineω-transaminases (e.g., SEQ ID NO: 11).

The reversible ω-transaminase from Chromobacterium violaceum (GenbankAccession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogousactivity accepting 7-aminoheptanoic acid as amino donor, thus formingthe first terminal amine group in pimelate semialdehyde (Kaulmann etal., Enzyme and Microbial Technology, 2007, 41, 628-637).

The reversible 4-aminobubyrate:2-oxoadipate transaminase fromStreptomyces griseus has demonstrated activity for the conversion of7-aminoheptanoate to pimelate semialdehyde (Yonaha et al., Eur. J.Biochem., 1985, 146, 101-106).

The reversible 5-aminovalerate transaminase from Clostridium viride hasdemonstrated activity for the conversion of 7-aminoheptanoate topimelate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19),8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed in7-aminoheptanal by a diamine transaminase classified, for example, underEC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as thegene product of YgjG from E. coli (Genbank Accession No. AAA57874.1, SEQID NO: 12). The transaminases set forth in SEQ ID NOs:7-10 and 11 alsocan be used to produce heptamethylenediamine. See, FIG. 7.

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine transaminase from E.coli strain B has demonstrated activityfor 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3),783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by adeacetylase classified, for example, under EC 3.5.1.62 such as anacetylputrescine deacetylase. The acetylputrescine deacetylase fromMicrococcus luteus K-11 accepts a broad range of carbon chain lengthsubstrates, such as acetylputrescine, acetylcadaverine andN8_acetylspermidine (see, for example, Suzuki et al., 1986, BBA-GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of1,7 Heptanediol

As depicted in FIG. 6, the terminal hydroxyl group can be enzymaticallyformed using a polypeptide having the activity of an alcoholdehydrogenase. For example, the second terminal hydroxyl group leadingto the synthesis of 1,7 heptanediol can be enzymatically formed in7-hydroxyheptanal by an alcohol dehydrogenase classified under EC1.1.1.- (e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184) such as thegene product of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1.

Enzymes generating N-acetyl-7-amino-3-oxoheptanoyl-CoA

As depicted in FIG. 1, N-acetyl-7-amino-oxoheptanoyl-CoA or a saltthereof can be biosynthesized from malonyl-CoA or L-aspartate throughthe intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA, which can beproduced from N-acetyl-5-aminopentanoyl-CoA using a polypeptide havingthe activity of a β-ketoacyl synthase, a β-ketothiolase, or aCoA-transferase. In some embodiments, a β-ketothiolase may be classifiedunder EC 2.3.1.16, such as the gene product of bktB or under EC2.3.1.174 such as the gene product of paaJ. In some embodiments, aβ-ketoacyl synthase may be classified under EC 2.3.1.180 such as thegene product of fabH, under EC 2.3.1.179 such as the gene product offabF or under EC 2.3.1.41 such as the gene product of fabB. In someembodiments, a CoA-transferase may be classified under EC 2.8.3- such asthe gene product of abfT.

Biochemical Pathways Pathways to 7-aminoheptanoate

In some embodiments, N-acetyl-5-amino-3-oxopentanoyl-CoA or a saltthereof is synthesized from the central metabolite, malonyl-CoA, byconversion of malonyl-CoA to malonate semialdehyde by a polypeptidehaving the activity of a malonyl CoA reductase classified, for example,under EC 1.2.1.75; followed by conversion of malonate semialdehyde toβ-alanine by a polypeptide having the activity of a β-alanine-pyruvateaminotransferase classified, for example, under EC 2.6.1.18; followed byconversion of β-alanine to N-acetyl-3-aminopropanoate by a polypeptidehaving the activity of an N-acetyl transferase classified, for example,under EC 2.3.1.13, EC 2.3.1.17 or EC 2.3.1.32; followed by conversion ofN-acetyl-3-aminopropanoate to N-acetyl-3-aminopropanoyl-CoA by apolypeptide having the activity of a CoA transferase classified, forexample, under EC 2.8.3.- or a polypeptide having the activity of a CoAligase classified, for example, under EC 6.2.1.-; followed by conversionof N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoAby a polypeptide having the activity of a β-ketoacyl synthase classifiedunder EC. 2.3.1.- (e.g., EC 2.3.1.180) or a polypeptide having theactivity of a β-ketothiolase classified under EC. 2.3.1.- (e.g.,EC2.3.1.16 or EC 2.3.1.174).

In some embodiments, N-acetyl-5-amino-3-oxopentanoyl-CoA or a saltthereof is synthesized from the central metabolite, L-aspartate, byconversion of L-aspartate to β-alanine by a polypeptide having theactivity of an aspartate α-decarboxylase classified, for example, underEC 4.1.1.11; followed by conversion of β-alanine toN-acetyl-3-aminopropanoate by a polypeptide having the activity of anN-acetyl transferase classified, for example, under EC 2.3.1.13, EC2.3.1.17 or EC 2.3.1.32; followed by conversion ofN-acetyl-3-aminopropanoate to N-acetyl-3-aminopropanoyl-CoA by apolypeptide having the activity of a CoA transferase classified, forexample, under EC 2.8.3.- or a polypeptide having the activity of a CoAligase classified, for example, under EC 6.2.1.-; followed by conversionof N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoAby a polypeptide having the activity of a β-ketoacyl synthaseclassified, for example, under EC 2.3.1.180 such as the gene product offabH or by a polypeptide having the activity of a β-ketothiolaseclassified, for example, under EC 2.3.1.16 such as the gene product ofbktB or under EC 2.3.1.174 such as the gene product of paaJ.

The intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereofis converted to N-acetyl-5-amino-3-hydroxypentanoyl-CoA by a polypeptidehaving the activity of a 3-hydroxyacyl-CoA dehydrogenase classified, forexample, under EC 1.1.1.- such as EC 1.1.1.35 (e.g., the gene product offadB), EC 1.1.1.36 (e.g., the gene product of phaB), or EC 1.1.1.157(e.g., the gene product of hbd); followed by conversion ofN-acetyl-5-amino-3-hydroxypentanoyl-CoA toN-acetyl-5-amino-pent-2-enoyl-CoA using a polypeptide having theactivity of an enoyl-CoA hydratase classified under, for example, EC4.2.1.17 such as the gene product of crt or under EC 4.2.1.119 such asthe gene product of phaJ; followed by conversion ofN-acetyl-5-amino-pent-2-enoyl-CoA to N-acetyl-5-amino-pentanoyl-CoA by apolypeptide having the activity of a trans-2-enoyl-CoA reductaseclassified under EC 1.3.1.38 or EC 1.3.1.44, such as the gene product ofter (Nishimaki et al., J. Biochem., 1984, 95:1315-1321; Shen et al.,2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012,51:6827-6837) or EC 1.3.1.8 (Inui et al., Eur. J. Biochem., 1984, 142,121-126); followed by conversion of N-acetyl-5-amino-pentanoyl-CoA toN-acetyl-7-amino-3-oxoheptanoyl-CoA by a polypeptide having the activityof a β-ketothiolase classified under, for example, EC 2.3.1.16 such asthe gene product of bktB or under EC 2.3.1.174 such as the gene productof paaJ; followed by conversion of N-acetyl-7-amino-3-oxoheptanoyl-CoAto N-acetyl-7-amino-3-hydroxyheptanoyl-CoA by a polypeptide having theactivity of a 3-hydroxyacyl-CoA dehydrogenase classified, for example,under EC 1.1.1.- such as EC 1.1.1.35 (e.g., the gene product of fadB),EC 1.1.1.36 (e.g., the gene product of phaB), or EC 1.1.1.157 (e.g., thegene product of hbd); followed by conversion ofN-acetyl-7-amino-3-hydroxyheptanoyl-CoA toN-acetyl-7-amino-hept-2-enoyl-CoA by a polypeptide having the activityof an enoyl-CoA-hydratase classified under, for example, EC 4.2.1.17such as the gene product of crt or under EC 4.2.1.119 such as the geneproduct of phaJ; followed by conversion of N-acetyl-7-amino-hept-2-enoyl-CoA to N-acetyl-7-aminoheptanoyl-CoA by apolypeptide having the activity of a trans-2-enoyl-CoA-reductaseclassified under EC 1.3.1.38 or EC 1.3.1.44, such as the gene product ofter (Nishimaki et al., J. Biochem., 1984, 95:1315-1321; Shen et al.,2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012,51:6827-6837) or EC 1.3.1.8 (Inui et al., Eur. J. Biochem., 1984, 142,121-126). See FIG. 1.

N-acetyl-7-aminoheptanoyl-CoA is then converted to 7-aminoheptanoate bya polypeptide having the activity of a thioesterase classified, forexample, under EC 3.1.2.-or a CoA-transferase classified, for example,under EC 2.8.3.- and subsequently a polypeptide having the activity of adeacetylase classified, for example, under EC 3.5.1.17 such as anacyl-lysine deacetylase from Achromobacter pestifer (see, for example,Chibate et al., 1970, Methods Enzymol., 19:756-762).See FIG. 2.

Pathways using 7-aminoheptanoate as central precursor to pimelic acid

In some embodiments, pimelic acid is synthesized from 7-aminoheptanoate,by conversion of 7-aminoheptanoate to pimelate semialdehyde by apolypeptide having the activity of a ω-transaminase classified, forexample, under EC 2.6.1.-, e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29,EC 2.6.1.48, or EC 2.6.1.82 such as that obtained from Chromobacteriumviolaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonasaeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonassyringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobactersphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 10), Vibriofluvialis (Genbank Accession No. AEA39183.1, SEQ ID NO: 12),Streptomyces griseus, or Clostridium viride. See, FIG. 3.

Pimelate semialdehyde is then converted to pimelic acid by a polypeptidehaving the activity of a dehydrogenase classified, for example, under EC1.2.1.- such as a 7-oxoheptanoate dehydrogenase (e.g., the gene productof ThnG), a 6-oxohexanoate dehydrogenase (e.g., the gene product ofChnE), a glutarate semialdehyde dehydrogenase classified, for example,under EC 1.2.1.20, a 5-oxopentanoate dehydrogenase such as the geneproduct of CpnE, or an aldehyde dehydrogenase classified under EC1.2.1.3. See FIG. 3.

Pathway using 7-aminoheptanoate as central precursor to7-hydroxyheptanoate

In some embodiments, 7-hydroxyheptanoate is synthesized from the centralprecursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate topimelate semialdehyde by a polypeptide having the activity of aω-transaminase classified, for example, under EC 2.6.1.-, e.g., EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asthat obtained from Chromobacterium violaceum (Genbank Accession No.AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (Genbank Accession No.AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (Genbank Accession No.AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (Genbank AccessionNo. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (Genbank Accession No.AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride;followed by conversion of pimelate semialdehyde to 7-hydroxyheptanoateby a polypeptide having the activity of an alcohol dehydrogenaseclassified, for example, under EC 1.1.1.2 such as the gene product ofYMR318C, a 6-hydroxyhexanoate dehydrogenase classified, for example,under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified, forexample, under EC 1.1.1.- such as the gene product of cpnD, or a4-hydroxybutanoate dehydrogenase classified, for example, under EC1.1.1.- such as the gene product of gabD. The alcohol dehydrogenaseencoded by YMR318C has broad substrate specificity, including theoxidation of C7 alcohols. See FIG. 5.

Pathway using 7-aminoheptanoate, 7-hydroxyheptanoate, pimelatesemialdehyde, or 1,7-heptanediol as a central precursor toheptamethylenediamine

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto 7-aminoheptanal by a polypeptide having the activity of a carboxylatereductase classified, for example, under EC 1.2.99.6 such as the geneproduct of car in combination with a phosphopantetheine transferaseenhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt genefrom Nocardia) or the gene products of GriC and GriD from Streptomycesgriseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed byconversion of 7-aminoheptanal to heptamethylenediamine by a polypeptidehaving the activity of a ω-transaminase such as a ω-transaminase in EC2.6.1.-, (e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.82 suchas SEQ ID NOs:7-12). The carboxylate reductase can be obtained, forexample, from Mycobacterium marinum (Genbank Accession No. ACC40567.1,SEQ ID NO: 2), Mycobacterium smegmatis (Genbank Accession No.ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus (Genbank Accession No.EFV11917.1, SEQ ID NO: 4), Mycobacterium massiliense (Genbank AccessionNo. EIV11143.1, SEQ ID NO: 5), Segniliparus rotundus (Genbank AccessionNo. ADG98140.1, SEQ ID NO: 6) or Mycobacterium smegmatis (GenbankAccession No. ABK75684.1, SEQ ID NO: 15). See FIG. 4.

The carboxylate reductase encoded by the gene product of car andenhancer npt or sfp has broad substrate specificity, including terminaldifunctional C4 and C5 carboxylic acids (Venkitasubramanian et al.,Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-hydroxyheptanoate (which can be produced asdescribed in FIGS. 1, 2 and 5), by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD (Suzuki et al., 2007, supra);followed by conversion of 7-aminoheptanal to 7-aminoheptanol by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12, seeabove; followed by conversion to 7-aminoheptanal by an alcoholdehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion toheptamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas SEQ ID NOs:7-12, see above. See FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto N7-acetyl-7-aminoheptanoate by a polypeptide having the activity ofan N-acetyltransferase such as a lysine N-acetyltransferase classified,for example, under EC 2.3.1.32; followed by conversion toN7-acetyl-7-aminoheptanal by a polypeptide having the activity of acarboxylate reductase classified, for example, under EC 1.2.99.6 such asthe gene product of car (see above, e.g., SEQ ID NO: 4, 5, or 6) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD; followed by conversion toN7-acetyl-1,7-diaminoheptane by a polypeptide having the activity of aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12, seeabove; followed by conversion to heptamethylenediamine by a polypeptidehaving the activity of a deacetylase classified, for example, under EC3.5.1.62 such as an acetylputrescine deacetylase. The acetylputrescinedeacetylase from Micrococcus luteus K-11 accepts a broad range of carbonchain length substrates, such as acetylputrescine, acetylcadaverine andN8_acetylspermidine (see, for example, Suzuki et al., 1986, BBA-GeneralSubjects, 882(1):140-142).See, FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, pimelate semialdehyde, by conversion of pimelatesemialdehyde to heptanedial by a polypeptide having the activity of acarboxylate reductase classified, for example, under EC 1.2.99.6 such asthe gene product of car (see above, e.g., SEQ ID NO:6) in combinationwith a phosphopantetheine transferase enhancer (e.g., encoded by a sfpgene from Bacillus subtilis or npt gene from Nocardia) or the geneproduct of GriC & GriD; followed by conversion to 7-aminoheptanal by apolypeptide having the activity of a ω-transaminase classified, forexample, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC2.6.1.82; followed by conversion to heptamethylenediamine by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12. SeeFIG. 4.

In some embodiments, heptamethylenediamine is synthesized from1,7-heptanediol by conversion of 1,7-heptanediol to 7-hydroxyheptanalusing a polypeptide having the activity of an alcohol dehydrogenaseclassified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2,EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C orYqhD or the protein having GenBank Accession No. CAA81612.1; followed byconversion to 7-aminoheptanol by a polypeptide having the activity of aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ IDNOs:7-12;followed by conversion to 7-aminoheptanal by a polypeptidehaving the activity of an alcohol dehydrogenase classified, for example,under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC1.1.1.184) such as the gene product of YMR318C or YqhD or the proteinhaving GenBank Accession No. CAA81612.1, followed by conversion toheptamethylenediamine by a polypeptide having the activity of aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs: 7-12. SeeFIG. 4.

Pathways using 7-hydroxyheptanoate as central precursor to1,7-heptanediol

In some embodiments, 1,7 heptanediol is synthesized from the centralprecursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a polypeptide having the activity of a carboxylatereductase classified, for example, under EC 1.2.99.6 such as the geneproduct of car (see above, e.g., SEQ ID NO: 2, 3, 4, 5, or 6) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus (Suzukiet al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of7-hydroxyheptanal to 1,7 heptanediol by a polypeptide having theactivity of an alcohol dehydrogenase (classified, for example, under EC1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184)such as the gene product of YMR318C or YqhD (from E. coli, GenBankAccession No. AAA69178.1) (see, e.g., Liu et al., Microbiology, 2009,155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; orJarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or theprotein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See, FIG. 6.

Cultivation Strategy

In some embodiments, one or more C7 building blocks are biosynthesizedin a recombinant microorganism using anaerobic, aerobic or micro-aerobiccultivation conditions. In some embodiments, the cultivation strategyentails nutrient limitation such as nitrogen, phosphate or oxygenlimitation.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes can be employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C7 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcycloheptane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcycloheptane processes has been demonstrated for numerousmicroorganisms, such as Delftia acidovorans and Cupriavidus necator(Ramsay et al., Applied and Environmental Microbiology, 1986,52(1):152-156).

In some embodiments, the microorganism is a prokaryote. For example, theprokaryote can be a bacterium from the genus Escherichia such asEscherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant microorganismsdescribed herein that are capable ofproducing one or more C7 building blocks.

In some embodiments, the microorganism is a eukaryote. For example, theeukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant microorganisms described herein that are capable ofproducing one or more C7 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the onlystep can be any one of the steps listed.

Furthermore, recombinant microorganisms described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant microorganism. Thisdocument provides microorganism cells of any of the genera and specieslisted and genetically engineered to express one or more (e.g., two,three, four, five, six, seven, eight, nine, 10, 11, 12 or more)recombinant forms of any of the enzymes recited in the document. Thus,for example, the microorganism cells can contain exogenous nucleic acidsencoding enzymes catalyzing one or more of the steps of any of thepathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined here can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C7 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C7 building block.

In some embodiments, the microorganism's tolerance to highconcentrations of a C7 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the microorganism's endogenous biochemical networkcan be attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA and β-alanine, (2) create an NADH or NADPHimbalance that may only be balanced via the formation of one or more C7building blocks, (3) prevent degradation of central metabolites, centralprecursors leading to and including one or more C7 building blocksand/or (4) ensure efficient efflux from the cell.

In some embodiments requiring intracellular availability of acetyl-CoAfor C7 building block synthesis, endogenous enzymes catalyzing thehydrolysis of acetyl-CoA such as short-chain length thioesterases can beattenuated in the microorganism.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenousphosphotransacetylase generating acetate such as pta can be attenuated(Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenous gene in anacetate synthesis pathway encoding an acetate kinase, such as ack, canbe attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as lactate dehydrogenase encoded by ldhA can be attenuated (Shen etal., 2011, supra).

In some embodiments, enzymes that catalyze anapleurotic reactions suchas PEP carboxylase and/or pyruvate carboxylase can be overexpressed inthe microorganism.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, endogenous genesencoding enzymes, such as menaquinol-fumarate oxidoreductase, thatcatalyze the degradation of phosphoenolpyruvate to succinate such asfrdBC can be attenuated (see, e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the alcohol dehydrogenase encoded by adhE can beattenuated (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C7building block synthesis, a recombinant formate dehydrogenase gene canbe overexpressed in the microorganism (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C7building block synthesis, a recombinant NADH-consuming transhydrogenasecan be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as pyruvatedecarboxylase can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, a recombinant acetyl-CoAsynthetase such as the gene product of acs can be overexpressed in themicroorganism (Satoh et al., J. Bioscience and Bioengineering, 2003,95(4):335-341).

In some embodiments, carbon flux can be directed into the pentosephosphate cycle to increase the supply of NADPH by attenuating anendogenous glucose-6-phosphate isomerase (EC 5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentosephosphate cycle to increase the supply of NADPH by overexpression a6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al.,2003, Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a gene such as UdhA encoding apuridine nucleotide transhydrogenase can be overexpressed in themicroorganisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012,Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 Building Block, a recombinantglyceraldehyde-3-phosphate-dehydrogenase gene such as GapN can beoverexpressed in the microorganisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant malic enzyme genesuch as maeA or maeB can be overexpressed in the microorganisms (Brighamet al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant glucose-6-phosphatedehydrogenase gene such as zwf can be overexpressed in themicroorganisms (Lim et al., J. Bioscience and Bioengineering, 2002,93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant fructose 1,6diphosphatase gene such as fbp can be overexpressed in themicroorganisms(Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, endogenous triose phosphateisomerase (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C7 building block, a recombinant glucosedehydrogenase such as the gene product of gdh can be overexpressed inthe microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003,95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH can be attenuated, such as the NADH generation cycle thatmay be generated via inter-conversion of glutamate dehydrogenasesclassified under EC 1.4.1.2 (NADH- specific) and EC 1.4.1.4(NADPH-specific).

In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3)that utilizes both NADH and NADPH as co-factors can be attenuated.

In some embodiments, a membrane-bound cytochrome P450 such as CYP4F3Bcan be solubilized by only expressing the cytosolic domain and not theN-terminal region that anchors the P450 to the endoplasmic reticulum(Scheller et al., J. Biol. Chem., 1994, 269(17):12779-12783).

In some embodiments, an enoyl-CoA reductase can be solubilized viaexpression as a fusion protein with a small soluble protein, forexample, the maltose binding protein (Gloerich et al., FEBS Letters,2006, 580, 2092-2096).

In some embodiments using microorganisms that naturally accumulatepolyhydroxyalkanoates, the endogenous polymer synthase enzymes can beattenuated in the microorganism strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed inthe microorganism to regenerate L-alanine from pyruvate as an aminodonor for ω-transaminase reactions.

In some embodiments, a L-glutamate dehydrogenase, a L-glutaminesynthetase, or a alpha-aminotransaminase can be overexpressed in themicroorganism to regenerate L-glutamate from 2-oxoglutarate as an aminodonor for ω-transaminase reactions.

In some embodiments, enzymes such as a pimeloyl-CoA dehydrogenaseclassified under, EC 1.3.1.62; an acyl-CoA dehydrogenase classified, forexample, under EC 1.3.8.7, EC 1.3.8.1, or EC 1.3.99.-; and/or abutyryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 thatdegrade central metabolites and central precursors leading to andincluding C7 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C7 building blocksvia Coenzyme A esterification such as CoA-ligases (e.g., an adipyl-CoAsynthetase) classified under, for example, EC 6.2.1.- can be attenuated.

In some embodiments, the efflux of a C7 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C7 building block.

The efflux of heptamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bltfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992,Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 7-aminoheptanoate and heptamethylenediamine can beenhanced or amplified by overexpres sing the solute transporters such asthe lysE transporter from Corynebacterium glutamicum (Bellmann et al.,2001, Microbiology, 147, 1765-1774).

The efflux of pimelic acid can be enhanced or amplified byoverexpressing a dicarboxylate transporter such as the SucE transporterfrom Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. &Biotech., 89(2), 327-335).

Producing C7 Building Blocks Using a Recombinant Microorganism

Typically, one or more C7 building blocks can be produced by providing amicroorganism and culturing the provided microorganism with a culturemedium containing a suitable carbon source as described above. Ingeneral, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C7 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C7 building block. Once produced, any method can be usedto isolate C7 building blocks. For example, C7 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of pimelic acid and 7-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofheptamethylenediamine and 1,7-heptanediol, distillation may be employedto achieve the desired product purity.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Enzyme activity of ω-transaminase using pimelatesemialdehyde as substrate and forming 7-aminoheptanoate

A nucleotide sequence encoding a N-terminal His-tag was added to thenucleic acid sequences from Chromobacterium violaceum, Pseudomonassyringae, Rhodobacter sphaeroides, and Vibrio fluvialis encoding theω-transaminases of SEQ ID NOs: 7, 9, 10 and 12, respectively (see FIG.7) such that N-terminal His-tagged ω-transaminases could be produced.Each of the resulting modified genes was cloned into a pET21a expressionvector under control of the T7 promoter and each expression vector wastransformed into a BL21[DE3] E. coli strain. The resulting recombinantE. coli strains were cultivated at 37° C. in a 250 mL shake flaskculture containing 50 mL LB media and antibiotic selection pressure,with shaking at 230 rpm. Each culture was induced overnight at 16° C.using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoateto pimelate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate,10 mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanoate and incubated at 25° C. for 4 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 7-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine. See FIG. 13. The gene productof SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12 accepted7-aminoheptanote as substrate as confirmed against the empty vectorcontrol. See FIG. 14.

Enzyme activity in the forward direction (i.e., pimelate semialdehyde to7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO 9,SEQ ID NO 10 and SEQ ID NO 12. Enzyme activity assays were performed ina buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing thepimelate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12 acceptedpimelate semialdehyde as substrate as confirmed against the empty vectorcontrol. See FIG. 15. The reversibility of the ω-transaminase activitywas confirmed, demonstrating that the ω-transaminases of SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted pimelatesemialdehyde as substrate and synthesized 7-aminoheptanoate as areaction product.

Example 2 Enzyme Activity of Carboxylate Reductase Using Pimelate asSubstrate and Forming Pimelate Semialdehyde

A nucleotide sequence encoding a HIS-tag was added to the nucleic acidsequences from Segniliparus rugosus and Segniliparus rotundus thatencode the carboxylate reductases of SEQ ID NOs: 4 (EFV11917.1) and 6(ADG98140.1), respectively (see FIG. 7), such that N-terminal HIS taggedcarboxylate reductases could be produced. Each of the modified genes wascloned into a pET Duet expression vector along with a sfp gene encodinga HIS-tagged phosphopantetheine transferase from Bacillus subtilis, bothunder the T7 promoter. Each expression vector was transformed into aBL21[DE3] E. coli strain and the resulting recombinant E. coli strainswere cultivated at 37° C. in a 250 mL shake flask culture containing 50mL LB media and antibiotic selection pressure, with shaking at 230 rpm.Each culture was induced overnight at 37° C. using an auto-inductionmedia.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde)were performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mMMgCl₂, 1 mM ATP and 1 mM NADPH. Each enzyme activity assay reaction wasinitiated by adding purified carboxylate reductase andphosphopantetheine transferase gene products or the empty vector controlto the assay buffer containing the pimelate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without pimelatedemonstrated low base line consumption of NADPH. See bars for EFV11917.1and ADG98140.1 in FIG. 8.

The gene products of SEQ ID NO: 4 (EFV11917.1) and SEQ ID NO: 6(ADG98140.1), enhanced by the gene product of sfp, accepted pimelate assubstrate, as confirmed against the empty vector control (see FIG. 9),and synthesized pimelate semialdehyde.

Example 3 Enzyme activity of carboxylate reductase using7-hydroxyheptanoate as substrate and forming 7-hydroxyheptanal

A nucleotide sequence encoding a His-tag was added to the nucleic acidsfrom Mycobacterium marinum, Mycobacterium smegmatis, Segniliparusrugosus, Mycobacterium smegmatis, Mycobacterium massiliense, andMycobacterium smegmatis that encode the carboxylate reductases of SEQ IDNOs: 2-6 and 15, respectively (GenBank Accession Nos. ACC40567.1,ABK71854.1, EFV11917.1, EIV11143.1, ADG98140.1, and ABK75684.1,respectively) (see FIG. 7) such that N-terminal HIS tagged carboxylatereductases could be produced. Each of the modified genes was cloned intoa pET Duet expression vector alongside a sfp gene encoding a His-taggedphosphopantetheine transferase from Bacillus subtilis, both undercontrol of the T7 promoter. Each expression vector was transformed intoa BL21[DE3] E. coli strain along with the expression vectors fromExample 3. Each resulting recombinant E. coli strain was cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 7-hydroxyheptanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without7-hydroxyheptanoate demonstrated low base line consumption of NADPH. SeeFIG. 8.

The gene products of SEQ ID NO 2-6 and 15, enhanced by the gene productof sfp, accepted 7-hydroxyheptanoate as substrate as confirmed againstthe empty vector control (see FIG. 10), and synthesized7-hydroxyheptanal.

Example 4 Enzyme activity of ω-transaminase for 7-aminoheptanol, forming7-oxoheptanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas syringae and Rhodobactersphaeroides nucleic acids encoding the ω-transaminases of SEQ ID NOs: 7,9 and 10, respectively (see FIG. 7) such that N-terminal HIS taggedω-transaminases could be produced. The modified genes were cloned into apET21a expression vector under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli strain. Each resultingrecombinant E. coli strain were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanolto 7-oxoheptanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 7-aminoheptanol had low base lineconversion of pyruvate to L-alanine. See FIG. 13.

The gene products of SEQ ID NOs: 7, 9 & 10 accepted 7-aminoheptanol assubstrate as confirmed against the empty vector control (see FIG. 18)and synthesized 7-oxoheptanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID Nos: 7, 9 & 10 accept7-oxoheptanol as substrate and form 7-aminoheptanol.

Example 5 Enzyme activity of -ωtransaminase using heptamethylenediamineas substrate and forming 7-aminoheptanal

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis nucleicacids encoding the ω-transaminases of SEQ ID NOs: 7-12, respectively(see FIG. 7) such that N-terminal HIS tagged ω-transaminases could beproduced. The modified genes were cloned into a pET21a expression vectorunder the T7 promoter. Each expression vector was transformed into aBL21[DE3] E. coli strain. Each resulting recombinant E. coli strain werecultivated at 37° C. in a 250 mL shake flask culture containing 50 mL LBmedia and antibiotic selection pressure, with shaking at 230 rpm. Eachculture was induced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,heptamethylenediamine to 7-aminoheptanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMheptamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′phosphate. Each enzyme activity assay reaction was initiated by addingcell free extract of the ω-transaminase gene product or the empty vectorcontrol to the assay buffer containing the heptamethylenediamine andthen incubated at 25° C. for 4 h, with shaking at 250 rpm. The formationof L-alanine was quantified via RP-HPLC.

Each enzyme only control without heptamethylenediamine had low base lineconversion of pyruvate to L-alanine. See FIG. 13.

The gene products of SEQ ID NOs: 7-12 accepted heptamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 16)and synthesized 7-aminoheptanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID NOs: 7-12 accept7-aminoheptanal as substrate and form heptamethylenediamine.

Example 6 Enzyme activity of carboxylate reductase forN7-acetyl-7-aminoheptanoate, forming N7-acetyl-7-aminoheptanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 3, 5, and 6 (see Examples 2 and 3, and FIG. 7) forconverting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal wasassayed in triplicate in a buffer composed of a final concentration of50 mM HEPES buffer (pH=7.5), 2 mM N7-acetyl-7-aminoheptanoate, 10 mMMgCl₂, 1 mM ATP, and 1 mM NADPH. The assays were initiated by addingpurified carboxylate reductase and phosphopantetheine transferase or theempty vector control to the assay buffer containing theN7-acetyl-7-aminoheptanoate then incubated at room temperature for 20min. The consumption of NADPH was monitored by absorbance at 340 nm.Each enzyme only control without N7-acetyl-7-aminoheptanoatedemonstrated low base line consumption of NADPH. See FIG. 8.

The gene products of SEQ ID NO 3, 5, and 6, enhanced by the gene productof sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmedagainst the empty vector control (see FIG. 11), and synthesizedN7-acetyl-7-aminoheptanal.

Example 7 Enzyme activity of ω-transaminase usingN7-acetyl-1,7-diaminoheptane, and forming N7-acetyl-7-aminoheptanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:7-12 (see Example 5, and FIG. 7) for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN7-acetyl-1,7-diaminoheptane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N7-acetyl-1,7-diaminoheptanedemonstrated low base line conversion of pyruvate to L-alanine. See FIG.13.

The gene product of SEQ ID NOs: 7-12 acceptedN7-acetyl-1,7-diaminoheptane as substrate as confirmed against the emptyvector control (see FIG. 17) and synthesized N7-acetyl-7-aminoheptanalas reaction product.

Given the reversibility of the ω-transaminase activity (see Example 1),the gene products of SEQ ID NOs: 7-12 accept N7-acetyl-7-aminoheptanalas substrate forming N7-acetyl-1,7-diaminoheptane.

Example 8 Enzyme activity of carboxylate reductase using pimelatesemialdehyde as substrate and forming heptanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO: 6 (seeExample 3 and FIG. 7) was assayed using pimelate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM pimelate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the pimelate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout pimelate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 8.

The gene product of SEQ ID N: 6, enhanced by the gene product of sfp,accepted pimelate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 12) and synthesized heptanedial.

Example 9 Enzyme activity of 4-hydroxybuterate-CoA transferase using5-ethanamidopentanoic acid as substrate and forming5-ethanamidopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA

A nucleotide sequence encoding a His-tag was added to the nucleic acidsequences from Cupriavidus necator, Clostridium propionicum, Clostridiumaminobutyricum, Citrobacter sp. A1, Acetobacter aceti, and E. coli K12encoding, in sequential order, the β-ketothiolase, priopionateCoA-transferase, 4-hydroxybuterate-CoA transferase. acetyl-CoAhydrolase, succinyl-CoA: acetate CoA-transferase, and thiolase of SEQ IDNOs: 16, 17, 18, 19, 20, and 21, respectively (see FIG. 7) forproduction of His-tagged versions of each protein. Each of the resultingmodified genes was cloned into a pET21a expression vector under controlof the T7 promoter and each expression vector was transformed into aBL21[DE3] E. coli strain. The resulting recombinant E. coli strains werecultivated at 37° C. in a 500 mL shake flask culture containing 100 mLLB media and antibiotic selection pressure, with shaking at 230 rpm.Each culture was induced overnight at 20° C. using 0.5 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation and passage through a 0.45 μm filter. Each of theHis-tagged proteins was purified from the supernatant by Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated by centrifugal filtration with a cut-off of 10 kD.

Enzyme assays were performed in two reactions for each of thesubstrates, N-acetyl-β-alanine (AC5) and 5-ethanamidopentanoic acid(AC7): reaction 1 and reaction 2 (see FIG. 19 for reaction schematic).

For reaction 1, each enzyme activity assay was performed in a buffercomposed of a final concentration of 25 mM HEPES buffer (pH=7.5), 50 mMN-acetyl-β-alanine (AC5) or 50 mM 5-ethanamidopentanoic acic (AC7), and2 mM acetyl CoA. Each enzyme activity assay was initiated by addingHis-tag purified enzymes or the empty vector control to the assay buffercontaining either the 50 mM N-acetyl-β-alanine or 5-ethanamidopentanoicacid and incubated at 37° C. for 2 h. The formation of5-ethanamidopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA wasmonitored by LC-MS to identify products by expected masses at distinctretention times.

For reaction 2, each enzyme activity assay was performed in a buffercomposed of a final concentration of 25 mM HEPES buffer (pH=7.5), 13 mMN-acetyl-β-alanyl-CoA (AC5-CoA) or 2.1 mM 5-ethanamidopentanoyl-CoA(AC7-CoA), and 5 mM acetyl CoA. Each enzyme activity assay was initiatedby adding His-tag purified enzymes or the empty vector control to theassay buffer containing either the 50 mM N-acetyl-β-alanine or5-ethanamidopentanoic acid (AC7-CoA) and incubated at 37° C. for 2 h.The formation of 7-ethanamido-3-oxoheptanoyl-CoA was monitored by LC-MSto identify products by expected masses at distinct retention times.

The 4-hydroxybuterate-CoA transferase gene product of SEQ ID NO: 18accepted 5-ethanamidopentanoic acid and 5-ethanamidopentanoyl-CoA assubstrate and formed 5-ethanamidopentanoyl-CoA and7-ethanamido-3-oxoheptanoyl-CoA as products, which was confirmed againstthe empty vector control. See row for EC 2.8.3- in Table 1, and LC-MSmass peaks confirming product identity by expected mass in FIG. 21(5-ethanamidopentanoyl-CoA: ESI MS expected [M+H]⁺=909.2017 and[M+2H]⁺²=445.1044; found 909.2017 and 445.1042) and FIG. 23(7-ethanamido-3-oxoheptanoyl-CoA: ESI MS expected [M+H]⁺=951.2120 and[M+2H]⁺²=476.1097; found 951.2132 and 476.1091).

Table 1 below presents the results of the enzyme assays. The enzymes arelisted by EC number, gene encoding the enzyme, and name. The enzymeassays were performed with acetyl-β-alanine (AC5) and5-ethanamidopentanoic acid (AC7) substrates in a sequence of tworeactions (see FIG. 19). Assays were monitored by LC-MS, and observedproduct (indicated by a check mark) and no product observed (indicatedby x), are reported in Table 1 for the 4-hydroxybutyrate-CoAtransferase.

AC5 AC7 Product Product Product Product Reaction Reaction ReactionReaction E.C. Gene Name 1 2 1 2 2.3.1.16/ Q0KBP1 BktB x x x x 2.3.1.92.8.3.8 Q9L3F7 237 x x x x 2.8.3- Q9RM86 244 x (?*) x (?*) ✓ ✓ 2.8.3.10J1G510 337 x x x x 2.8.3.18 B3EY95 344 x x x x 2.3.1.174 P0C7L2 PaaJ x xx x *(?): Product (f) and (e) may be present but not clear.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of producing N-acetyl-5-amino-3-oxopentanoyl-CoA or a saltthereof, said method comprising enzymatically convertingN-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA ora salt thereof using a polypeptide having the activity of a β-ketoacylsynthase or a β-ketothiolase classified under EC. 2.3.1.- and/or a CoAtransferase classified under EC 2.8.3.-.
 2. The method of claim 1,wherein said polypeptide having the activity of a β-ketoacyl synthase isclassified under EC 2.3.1.41, EC 2.3.1.179 or EC 2.3.1.180 and whereinsaid polypeptide having the activity of a β-ketothiolase is classifiedunder EC 2.3.1.16 or EC 2.3.1.174.
 3. The method of claim 1 any one ofclaims 1 2, wherein said polypeptide having the activity of aβ-ketothiolase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NOs: 1 or 13 and said polypeptide havingthe activity of a β-ketoacyl synthase has at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO: 14 and saidpolypeptide having the activity of a CoA transferase has at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.4. The method of claim 3, wherein said polypeptide having the activityof a β-ketothiolase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NOs: 1 or 13 and is capable of convertingN-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA andsaid polypeptide having the activity of a β-ketoacyl synthase has atleast 70% sequence identity to the amino acid sequence set forth in SEQID NO: 14 and is capable of converting N-acetyl-3-aminopropanoyl-CoA toN-acetyl-5-amino-3-oxopentanoyl-CoA and said polypeptide having theactivity of a CoA transferase has at least 70% sequence identity to theamino acid sequence set forth in SEQ ID NO: 19 and is capable ofconverting N-acetyl-3-aminopropanoyl-CoA toN-acetyl-5-amino-3-oxopentanoyl-CoA.
 5. The method of claim 1, furthercomprising enzymatically converting N-acetyl-5-amino-3-oxopentanoyl-CoAor the salt thereof to 7-aminoheptanoate using polypeptides having theenzymatic activities of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoAhydratase, a trans-2-enoyl-CoA reductase, a β-ketothiolase, athioesterase or a CoA transferase and a deacetylase. 6.-12. (canceled)13. A method for biosynthesizing 7-aminoheptanoate, said methodcomprising enzymatically synthesizingN-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof fromN-acetyl-3-aminopropanoyl-CoA using a polypeptide having the activity ofa β-ketoacyl synthase or a β-ketothiolase classified under EC. 2.3.1.-and/or a CoA transferase classified under EC 2.8.3-, and enzymaticallyconverting N-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof to7-aminoheptanoate.
 14. The method of claim 13, whereinN-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof is converted toN-acetyl-5-amino-3-hydroxypentanoyl-CoA using a polypeptide having theactivity of a 3-hydroxyacyl-CoA dehydrogenase;N-acetyl-5-amino-3-hydroxypentanoyl-CoA is converted to5-amino-pent-2-enoyl-CoA using polypeptide having the activity of anenoyl-CoA hydratase; N-acetyl-5-amino-pent-2-enoyl-CoA is converted toN-acetyl-5-amino-pentanoyl-CoA using a polypeptide having the activityof a trans-2-enoyl-CoA-reductase; N-acetyl-5-amino-pentanoyl-CoA isconverted to N-acetyl-7-amino-3-oxoheptanoyl-CoA using a polypeptidehaving the activity of a β-ketothiolase;N-acetyl-7-amino-3-oxoheptanoyl-CoA is converted toN-acetyl-7-amino-3-hydroxyheptanoyl-CoA using a polypeptide having theactivity of a 3-hydroxyacyl-CoA-dehydrogenase;N-acetyl-7-amino-3-hydroxyheptanoyl-CoA is converted toN-acetyl-7-amino-hept-2-enoyl-CoA using a polypeptide having theactivity of an enoyl-CoA hydratase; N-acetyl-7-amino-hept-2-enoyl-CoA isconverted to N-acetyl-7-aminoheptanoyl-CoA using a polypeptide havingthe activity of a trans-2-enoyl-CoA reductase;N-acetyl-7-aminoheptanoyl-CoA is converted to N-acetyl-7-aminoheptanoateusing a polypeptide having the activity of a thioesterase or a CoAtransferase; and N-acetyl-7-aminoheptanoate is converted to7-aminoheptanoate using a polypeptide having the activity of adeacetylase.
 15. The method of claim 5, said method further comprisingenzymatically converting 7-aminoheptanoate to pimelic acid,7-hydroxyheptanoate, heptamethylenediamine or 1,7-heptanediol or acorresponding salt thereof in one or more steps.
 16. The method of claim15, wherein 7-aminoheptanoate is converted to pimelic acid using one ormore polypeptides having the enzymatic activity of a ω-transaminase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a5-oxopentanoate dehydrogenase, or an aldehyde dehydrogenase.
 17. Themethod of claim 15, wherein 7-aminoheptanoate is converted to7-hydroxyheptanoate using one or more polypeptides having the enzymaticactivity of an alcohol dehydrogenase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoatedehydrogenase, and a ω-transaminase.
 18. The method of claim 15, wherein7-aminoheptanoate is converted to heptamethylenediamine usingpolypeptides having the enzymatic activity of a carboxylate reductaseand a ω-transaminase.
 19. The method of claim 15, wherein7-aminoheptanoate is converted to heptamethylenediamine usingpolypeptides having the enzymatic activity of a carboxylate reductase, aω-transaminase and an alcohol dehydrogenase.
 20. The method of claim 15,wherein 7-aminoheptanoate is converted to heptamethylenediamine usingpolypeptides having the enzymatic activity of an N-acetyltransferase, acarboxylate reductase, a ω-transaminase, and a deacetylase.
 21. Themethod of claim 15, wherein 7-aminoheptanoate is converted toheptamethylenediamine using polypeptides having the enzymatic activityof an alcohol dehydrogenase and a ω-transaminase.
 22. (canceled) 23.(canceled)
 24. The method of claim 15, wherein 7-hydroxyheptanoate isconverted to 1,7-heptanediol using a polypeptide having the activity ofa carboxylate reductase and a polypeptide having the activity of analcohol dehydrogenase.
 25. (canceled)
 26. (canceled)
 27. The method ofclaim 1, wherein said N-acetyl-3-aminopropanoyl-CoA is enzymaticallyproduced from malonyl-CoA or L-aspartate.
 28. The method of claim 27,wherein said N-acetyl-3-aminopropanoyl-CoA is enzymatically producedfrom malonyl-CoA or L-aspartate using one or more polypeptides havingthe enzymatic activity of a malonyl-CoA-reductase, a β-alanine-pyruvateaminotransferase, an α-aspartate decarboxylase, an N-acetyl transferase,a CoA transferase and a CoA Ligase.
 29. The method of claim 1, whereinsaid method is performed in a recombinant microorganism.
 30. The methodof claim 29, wherein said microorganism is subjected to a cultivationstrategy under aerobic, anaerobic or, micro-aerobic cultivationconditions.
 31. The method of claim 29, wherein said microorganism iscultured under conditions of nutrient limitation.
 32. (canceled)
 33. Themethod of claim 29, wherein the principal carbon source fed to thefermentation derives from a biological feedstock.
 34. (canceled)
 35. Themethod of claim 29, wherein the principal carbon source fed to thefermentation derives from a non-biological feedstock.
 36. (canceled) 37.The method of claim 29, wherein the microorganism is a prokaryote. 38.(canceled)
 39. (canceled)
 40. The method of claim 29, wherein themicroorganism is a eukaryote.
 41. (canceled)
 42. (canceled)
 43. Themethod of claim 29, wherein the microorganism's tolerance to highconcentrations of a C7 building block is improved relative to a wildtype organism.
 44. (canceled)
 45. The method of claim 29 , wherein saidmicroorganism comprises an attenuation to one or more of the followingenzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, an alcoholdehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvatedecarboxylase, a glucose-6-phosphate isomerase, NADH-consumingtranshydrogenase, an NADH-specific glutamate dehydrogenase, aNADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoAdehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocksand central precursors as substrates; a butaryl-CoA dehydrogenase; or anadipyl-CoA synthetase.
 46. The method of claim 29, wherein saidmicroorganism overexpresses one or more genes encoding: an acetyl-CoAsynthetase, a 6-phosphogluconate dehydrogenase; a transketolase; apuridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase;a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucosedehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase;a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutaminesynthetase; a diamine transporter, a dicarboxylate transporter, and/or amultidrug transporter.
 47. A recombinant microorganism comprising atleast one exogenous nucleic acid encoding a polypeptide having theenzymatic activity of (i) a β-ketoacyl synthase and/or a β-ketothiolase,(ii) a thioesterase or a CoA transferase, (iii) a deacetylase, and oneor more of (iv) 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoAhydratase, and (v) a trans-2-enoyl-CoA reductase, said microorganismproducing 7-aminoheptanoate or a corresponding salt thereof.
 48. Therecombinant microorganism of claim 47, said microorganism furthercomprising one or more of the following exogenous enzymes:ω-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, a 5-pentanoate dehydrogenase, or an aldehydedehydrogenase, said microorganism further producing pimelic acid or acorresponding salt thereof.
 49. The recombinant microorganism of claim47, said microorganism further comprising one or more of the followingexogenous enzymes: a ω-transaminase, a 6-hydroxyhexanoate dehydrogenase,a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase,and an alcohol dehydrogenase, said microorganism further producing7-hydroxyheptanoate or a corresponding salt thereof.
 50. The recombinantmicroorganism of claim 47, said microorganism further comprising one ormore of the following exogenous enzymes: a carboxylate reductase, aω-transaminase, a deacylase, a N-acetyl transferase, or an alcoholdehydrogenase, said microorganism further producingheptamethylenediamine or a corresponding salt thereof. 51.-54.(canceled)
 55. The recombinant microorganism of claim 47, saidmicroorganism further comprising an exogenous carboxylate reductase andan exogenous alcohol dehydrogenase, said microorganism further producing1,7-heptanediol or a corresponding salt thereof.
 56. The recombinantmicroorganism of claim 47, said microorganism further comprising one ormore of the following exogenous enzymes: an aspartate-α-decarboxylase; amalonyl-CoA reductase; a β-alanine-pyruvate-aminotransferase; anN-acetyl transferase; a thioesterase; a CoA-transferase; a CoA ligaseand a deacetylase.
 57. A non-naturally occurring microorganismcomprising at least one exogenous nucleic acid encoding at least onepolypeptide having the activity of at least one enzyme, at least onesubstrate and at least one product, depicted in any one of FIGS. 1 to 6.58. A nucleic acid construct or expression vector comprising apolynucleotide encoding a polypeptide having β-ketothiolase activity,wherein the polynucleotide is operably linked to one or moreheterologous control sequences that direct production of the polypeptideand wherein the polypeptide having β-ketothiolase activity is selectedfrom the group consisting of: (a) a polypeptide having at least 70%sequence identity to the polypeptide of SEQ ID NO: 1; and (b) apolypeptide having at least 70% sequence identity to the polypeptide ofSEQ ID NO:
 13. 59. A nucleic acid construct or expression vectorcomprising a polynucleotide encoding a polypeptide having β-ketoacylsynthase activity, wherein the polynucleotide is operably linked to oneor more heterologous control sequences that direct production of thepolypeptide and wherein the polypeptide having β-ketoacyl synthaseactivity has at least 70% sequence identity to the polypeptide of SEQ IDNO:
 14. 60. A nucleic acid construct or expression vector comprising apolynucleotide encoding a polypeptide having carboxylate reductaseactivity, wherein the polynucleotide is operably linked to one or moreheterologous control sequences that direct production of the polypeptideand wherein the polypeptide having carboxylate reductase activity isselected from the group consisting of: (a) a polypeptide having at least70% sequence identity to the polypeptide of SEQ ID NO: 2; (b) apolypeptide having at least 70% sequence identity to the polypeptide ofSEQ ID NO: 3; (c) a polypeptide having at least 70% sequence identity tothe polypeptide of SEQ ID NO: 4; (d) a polypeptide having at least 70%sequence identity to the polypeptide of SEQ ID NO: 5; (e) a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:6; and (f) a polypeptide having at least 70% sequence identity to thepolypeptide of SEQ ID NO:
 15. 61. A nucleic acid construct or expressionvector comprising a polynucleotide encoding a polypeptide havingω-transaminase activity, wherein the polynucleotide is operably linkedto one or more heterologous control sequences that direct production ofthe polypeptide and wherein the polypeptide having ω-transaminaseactivity is selected from the group consisting of: (a) a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:7; (b) a polypeptide having at least 70% sequence identity to thepolypeptide of SEQ ID NO: 8; (c) a polypeptide having at least 70%sequence identity to the polypeptide of SEQ ID NO: 9; (d) a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:10; (e) a polypeptide having at least 70% sequence identity to thepolypeptide of SEQ ID NO:11 and (f) a polypeptide having at least 70%sequence identity to the polypeptide of SEQ ID NO:12.
 62. (canceled) 63.(canceled)
 64. A non-naturally occurring biochemical network comprisingan N-acetyl-3-aminopropanoyl-CoA, an exogenous nucleic acid encoding apolypeptide having the activity of a β-ketothiolase or a β-ketoacylsynthase classified under EC. 2.3.1, and anN-acetyl-5-amino-3-oxopentanoyl-CoA.
 65. A non-naturally occurringbiochemical network comprising at least one exogenous nucleic acidencoding a polypeptide having the enzymatic activity of (i) a β-ketoacylsynthase and/or a β-ketothiolase, (ii) a thioesterase or a CoAtransferase, (iii) a deacetylase, and one or more of (iv)3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) atrans-2-enoyl-CoA reductase, said microorganism producing7-aminoheptanoate.
 66. (canceled)
 67. A bio-derived, bio-based orfermentation-derived product, wherein said product comprises: (i) acomposition comprising at least one bio-derived, bio-based orfermentation-derived compound according to claim 15 or any combinationthereof;(ii) a bio-derived, bio-based or fermentation-derived polymercomprising the bio-derived, bio-based or fermentation-derivedcomposition or compound of (i), or any combination thereof; (iii) abio-derived, bio-based or fermentation-derived resin comprising thebio-derived, bio-based or fermentation-derived compound or bio-derived,bio-based or fermentation-derived composition of (i) or any combinationthereof or the bio-derived, bio-based or fermentation-derived polymer of(ii) or any combination thereof; (iv) a molded substance obtained bymolding the bio-derived, bio-based or fermentation-derived polymer of(ii) or the bio-derived, bio-based or fermentation-derived resin of(iii), or any combination thereof; (v) a bio-derived, bio-based orfermentation-derived formulation comprising the bio-derived, bio-basedor fermentation-derived composition of (i), bio-derived, bio-based orfermentation-derived compound of (i), bio-derived, bio-based orfermentation-derived polymer of (ii), bio-derived, bio-based orfermentation-derived resin of (iii), or bio-derived, bio-based orfermentation-derived molded substance of (iv), or any combinationthereof; or (vi) a bio-derived, bio-based or fermentation-derivedsemi-solid or a non-semi-solid stream, comprising the bio-derived,bio-based or fermentation-derived composition of (i), bio-derived,bio-based or fermentation-derived compound of (i), bio-derived,bio-based or fermentation-derived polymer of (ii), bio-derived,bio-based or fermentation-derived resin of(iii), bio-derived, bio-basedor fermentation-derived formulation of (v), or bio-derived, bio-based orfermentation-derived molded substance of (iv), or any combinationthereof.
 68. A method of producing 7-ethanamido-3-oxoheptanoyl-CoA or asalt thereof, said method comprising enzymatically converting5-ethanamidopentanoic acid to 7-ethanamido-3-oxoheptanoyl-CoA or a saltthereof using a polypeptide having the activity of a β-ketoacyl synthaseor a β-ketothiolase classified under EC. 2.3.1.-, further comprisingenzymatically converting 5-ethanamidopentanoic acid or the salt thereofto 7-ethanamido-3-oxoheptanoyl-CoA using polypeptides having theenzymatic activities of a β-ketothiolase, CoA transferase, acetyl-CoAhydrolase, and thiolase.
 69. The method of claim 68, wherein saidCoA-transferase is classified under EC 2.8.3.-.
 70. A nucleic acidconstruct or expression vector comprising a polynucleotide encoding apolypeptide having CoA transferase activity, wherein the polynucleotideis operably linked to one or more heterologous control sequences thatdirect production of the polypeptide and wherein the polypeptide havingCoA transferase activity has at least 70% sequence identity to thepolypeptide of SEQ ID NO: 19.